Academic literature on the topic 'CAMP assay'
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Journal articles on the topic "CAMP assay"
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Golla, Rajasree, and Ramakrishna Seethala. "A Homogeneous Enzyme Fragment Complementation Cyclic AMP Screen for GPCR Agonists." Journal of Biomolecular Screening 7, no. 6 (December 2002): 515–25. http://dx.doi.org/10.1177/1087057102238625.
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In the new high-throughput screening (HTS) campaign, receptor functional assays, 3’,5’-cyclic adenosine mono-phosphate (cAMP), intracellular [Ca2+]i, phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter™ enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Gαs-coupled receptor. In the EFC-cAMP assay, the β-galactosidase (β-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the β-gal enzyme acceptor (EA) fragment to form an active β-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Gαs to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC50 of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC50 ~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 μM in suspension cells. The assay is very robust, with a Z value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
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Elster, Lisbeth, Christian Elling, and Anders Heding. "Bioluminescence Resonance Energy Transfer as a Screening Assay: Focus on Partial and Inverse Agonism." Journal of Biomolecular Screening 12, no. 1 (November 12, 2006): 41–49. http://dx.doi.org/10.1177/1087057106295895.
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The reported data for compound screening with the bioluminescence resonance energy transfer (BRET2) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven β2 adrenergic receptor (β2-AR) agonists were tested for full or partial agonism in an improved version of the receptor/β-arrestin2 BRET2 assay and in 2 cyclic adenosine monophosphate (cAMP) assays (column cAMP assay and ALPHAscreen™ cAMP assay). Tested in the highly sensitive ALPHAscreen™ cAMP assay, all selected agonists behaved as full agonists, using isoproterenol as a reference compound. In the less sensitive column cAMP assay, ephedrine and dopamine had a clear partial response. For the BRET2 assay, a highly graded picture was obtained. Moreover, β2-AR antagonists were tested for inverse agonism. Pronounced inverse agonism was detected in the ALPHAscreen™ cAMP assay. Only marginal inverse agonistic responses were seen for alprenolol and pindolol in the column cAMP assay, and no inverse agonism was seen in the BRET2 assay. For the β2-AR, the BRET2 assay may be superior for secondary screening of agonists where a separation of full and partial agonists is needed and the ALPHAscreen™ cAMP assay may be preferred for primary screening of agonists where all receptor activating compounds are desired.
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Chiulli, Anthony C., Karen Trompeter, and Michelle Palmer. "A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels." Journal of Biomolecular Screening 5, no. 4 (June 2000): 239–47. http://dx.doi.org/10.1177/108705710000500406.
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The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.
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Kool, Jeroen, André Van Marle, Saskia Hulscher, Maurice Selman, Dick J. Van Iperen, Klaas Van Altena, Michel Gillard, et al. "A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production." Journal of Biomolecular Screening 12, no. 8 (December 2007): 1074–83. http://dx.doi.org/10.1177/1087057107308881.
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A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z′ factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H3 G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC50 values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements. ( Journal of Biomolecular Screening 2007:1074-1083)
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Sugiyama, Atsushi, Phi Wiegn, Scott McKnite, and Keith G. Lurie. "Enzymatic fluorometric assay for tissue cAMP." Journal of Clinical Laboratory Analysis 8, no. 6 (1994): 437–42. http://dx.doi.org/10.1002/jcla.1860080616.
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Martikkala, Eija, Anita Rozwandowicz-Jansen, Pekka Hänninen, Ulla Petäjä-Repo, and Harri Härmä. "A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay." Journal of Biomolecular Screening 16, no. 3 (February 22, 2011): 356–62. http://dx.doi.org/10.1177/1087057110397356.
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G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.
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Prystay, Linda, Annie Gagne, Patricia Kasila, Li-An Yeh, and Peter Banks. "Homogeneous Cell-Based Fluorescence Polarization Assay for the Direct Detection of cAMP." Journal of Biomolecular Screening 6, no. 2 (April 2001): 75–82. http://dx.doi.org/10.1177/108705710100600203.
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A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2cr (CRF2a) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 μM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.
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Tian, Ling, Rongsheng E. Wang, Ying Fei, and Yie-Hwa Chang. "A Homogeneous Fluorescent Assay for cAMP-Phosphodiesterase Enzyme Activity." Journal of Biomolecular Screening 17, no. 3 (November 7, 2011): 409–14. http://dx.doi.org/10.1177/1087057111426901.
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Cyclic adenosine monophosphate–phosphodiesterases (cAMP-PDEs) regulate the cellular level of cAMP by selectively catalyzing the hydrolysis of the phosphodiester bond in the cAMP molecule. They play important roles in modulating cellular and physiological functions. There is a growing interest in the study of cAMP-PDEs as therapeutic targets. We describe a novel method for measuring the enzyme activity of cAMP-PDEs that is based on a homogeneous fluorescence assay employing a cAMP-dependent DNA-binding protein (CAP). We demonstrate that the assay is quick and robust compared to traditional methods and is expected to be cost-effective for high-throughput screening of cAMP-PDE inhibitors. The usefulness of the assay is demonstrated by measuring IC50 values of three nonselective PDE inhibitors and by kinetic measurements of cAMP-PDEs from various rat tissues.
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Diana, Tanja, Paul D. Olivo, Yie-Hwa Chang, Christian Wüster, Michael Kanitz, and George J. Kahaly. "Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies." European Thyroid Journal 9, no. 2 (November 27, 2019): 67–72. http://dx.doi.org/10.1159/000504509.
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Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves’ disease (GD). We tested a novel homogeneous fluorescent 3′,5′ cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. Results: Both cell lines were stimulated with forskolin concentrations (0.006–200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8–25 and 3.1–50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006–50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8–12.5 and 0.2–3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. Conclusion: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.
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Danish, Azeem, Robin Gedschold, Sonja Hinz, Anke C. Schiedel, Dominik Thimm, Peter Bedner, Christian Steinhäuser, and Christa E. Müller. "A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators." International Journal of Molecular Sciences 22, no. 3 (January 31, 2021): 1417. http://dx.doi.org/10.3390/ijms22031417.
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Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.
Dissertations / Theses on the topic "CAMP assay"
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Lupica, Samuel J. "Nitrate Toxicity to Common Carp Measured Noninvasively by Novel Enzyme-linked Immunosorbent Assay for Cortisol." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1226956326.
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Pandrangi, Raj Gopal. "Determination of genotoxic stress in feral populations of bullheads and carp using the alkaline single cell gel assay." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0022/NQ30295.pdf.
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Camara, Ramatoulie. "Design, synthesis and biological evaluation of potential inhibitors of S100P, a protein implicated in pancreatic cancer." Thesis, University of Hertfordshire, 2015. http://hdl.handle.net/2299/17117.
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Pancreatic cancer is relatively uncommon. Despite its relative scarcity, it is the fourth-ranked cancer killer in the Western world with less than a 5% 5-year survival rate. The high mortality rate is due to the asymptomatic nature of the disease and the advanced stage at which it is usually diagnosed. S100P is a calcium-binding protein that has been shown to be highly expressed in the early stages of pancreatic cancer and has been proposed as a potential therapeutic target via the blocking of its interaction with its receptor RAGE, the receptor for advanced glycation end-products. In this thesis, computational techniques were employed on the NMR ensemble of S100P (PDB Accession code 1OZO) to identify potential inhibitors of the S100P-RAGE interaction in the hope of identifying a series of novel leads that could be developed into clinical candidates for the treatment of pancreatic cancer. In silico studies identified putative binding sites at the S100P dimeric interface capable of accommodating cromolyn, an anti-allergy drug shown to bind to the protein both in vitro and in vivo. Virtual screening of >1 million lead-like compounds using 3D pharmacophore models derived from the predicted binding interactions between S100P and cromolyn, identified 9,408 'hits'. These were hierarchically clustered according to similarities between chemical structures into 299 clusters and 77 singletons. Biological screening of 17 of the 'hits' identified from virtual screening stuidies, 4 of which were synthesised in-house, against pancreatic cancer cell lines identified five compounds that demonstrated an equal or greater capacity to reduce BxPC-3 S100P-expressing pancreatic cells' metastatic potential in vitro relative to cromolyn. Compound 24 in particular, showed significant (p<0.05) inhibition of invasion of these cells at a concentration of 100 μM that was comparable to cromolyn at the same concentration. This compound, structurally distinct from cromolyn, was successfully synthesised, purified and characterised in-house alongside 39 of its analogues. Biological screening of compound 24 and four of its analogues for anti-proliferative activity against BxPC-3 and Panc-1 pancreatic cancer cell lines showed all five compounds significantly (p < 0.0001) inhibiting proliferation in both cell lines at a concentration of 1 μM relative to the non-treated control. Hence, structurally distinct compounds that show promising inhibitory activity on the metastasis and proliferation of pancreatic cancer cells have been identified using a structure-based drug design methodology. These compounds, with further optimisation, could provide good starting points as therapeutic lead candidates for the treatment of pancreatic cancer.
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Teixeira, Mirian Vieira. "Análise da expressão e das interações da subunidade catalítica da PKA do fungo patogênico Paracoccidioides ssp." Universidade Federal de Goiás, 2016. http://repositorio.bc.ufg.br/tede/handle/tede/5811.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The Paracoccidioides genus comprises a complex of pathogenic fungi that are the etiologic agents of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The infection begins after inhalation of fungal propagules, which reach the epithelium of the alveoli where the transition from the mycelial to the pathogenic yeast form. Host elevated temperature triggers the morphological switch, which is necessary for fungal pathogenicity. The cAMP/protein kinase A (PKA) signaling pathway has been shown to be important in controlling morphological changes and the pathogenicity of several pathogenic fungi. Evidence also highlights the importance of the cAMP/PKA pathway in the morphological transition of Paracoccidioides. PKA is the major effector of this signaling pathway. The protein is an inactive tetramer composed of regulatory subunit, encoded by the BCY1 gene; and catalytic subunit, encoded by the TPK2 gene. Upon binding of cAMP to the regulatory subunits, the catalytic subunits dissociate and become active. Activated PKA subsequently phosphorylates protein kinases, transcription factors, and other substrates to control several biological processes. In this study, we evaluated the expression and interactions of Tpk2 protein Paracoccididioides spp. The Tpk2 is present in mycelium decreased during the initial stages of transition phases, and increases again at the end of differentiation, with maximal levels in yeast. We analyzed the interactions of recombinant Tpk2p with Paracoccidioides proteins using pull-down assays followed by MS analysis. Two interacting proteins were identified: the heat shock protein 90 (Hsp90) and a conserved hypothetical protein with a MFS domain. Hsp90 is involved in the regulation of morphogenesis, development and virulence in several thermal dimorphic fungi. These data are important for understanding the mechanisms that trigger the transition phases in Paracoccidioides.
O gênero Paracoccidioides compreende um complexo de fungos patogênicos, que são os agentes etiológicos da paracoccidioidomicose (PCM), a micose sistêmica mais prevalente na América Latina. A infecção inicia-se com a inalação de propágulos do fungo, que atingem o epitélio dos alvéolos pulmonares, onde ocorre à transição da forma de micélio para a forma patogênica, de levedura. Há evidências de que a temperatura seja o principal fator responsável pela diferenciação celular desses fungos, e sua patogenicidade é frequentemente associada com a transição dimórfica. A via de sinalização cAMP/ proteína quinase A (PKA) controla alterações morfológicas e de virulência/patogenicidade em várias espécies de fungos patogênicos humanos. Evidências apontam também para a importância da via cAMP/PKA em Paracoccidioides spp. A PKA é o principal efetor desta via de sinalização. A proteína na forma inativa é um tetrâmero composto de subunidade regulatória, codificada pelo gene BCY1; e subunidade catalítica, codificada pelo gene TPK2. Após a ligação de cAMP às subunidades regulatórias, as subunidades catalíticas dissociam-se e tornam-se ativas. Ativada a PKA fosforila proteína-quinases, fatores de transcrição, e outros substratos para controlar diversos processos biológicos. Neste estudo, avaliamos a expressão e as interações da proteína Tpk2 de Paracoccididioides spp. A Tpk2 está presente em micélio, diminui nos estágios iniciais da transição de fases e volta a aumentar no final da diferenciação, apresentando níveis máximos na levedura. Foram analisadas as interações de Tpk2p recombinante com proteínas de Paracoccidioides utilizando ensaios de pull-down, seguido por análise de MS. Foram identificadas duas proteínas que interagem: a proteína de choque térmico 90 (Hsp90) e uma proteína hipotética conservada com um domínio MFS. Hsp90 está envolvido na regulação da morfogênese, desenvolvimento e virulência em vários fungos dimórficos térmicos. Estes dados são importantes para entendimento dos mecanismos que disparam a transição de fases em Paracoccidioides spp.
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Suntharalingam, Gaayathiri [Verfasser]. "Der Einfluss der Induktion von Tumornekrosefaktor α und Transforming-Growth-Factor β auf die epithelial-mesenchymale Transition oraler Plattenepithelkarzinome im CAM-Assay / Gaayathiri Suntharalingam." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1228364583/34.
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Leupold, Jörg. "Untersuchungen zur Regulation des Invasionsgens Urokinase-Rezeptor (CD87) durch den Tumorsuppressor Pdcd4 unter gleichzeitiger methodischer Neuetablierung eines quantitativen CAM-Assays." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-36138.
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Ruhe, Larissa. "Investigation of cap-independent translation initiation in neuronal differentiation." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21184.
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Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap-abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt.
Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt.
Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden.Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E‑BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap-independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.
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Amui, Saulo França. "Do laboratório ao campo virtual: desenvolvimento de um banco de dados de venenos de serpentes brasileiras e análise computacional de estruturas primárias de fosfolipases A2." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/60/60134/tde-21052007-094343/.
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Os avanços tecnológicos vêm contribuindo cada vez mais nas áreas biológicas e científicas oferecendo ferramentas computacionais e sistemas específicos que em análise de dados in silico fornecem resultados rápidos e confiáveis. O presente projeto propõe o desenvolvimento de um portal na Internet para instalação e utilização de um banco de dados laboratoriais das principais serpentes brasileiras com seus respectivos venenos e antivenenos naturais, e a análise dos dados obtidos em ensaios farmacológicos, e bioquímicos. Utilizando a via de comunicação e interação mais simples atualmente, a Internet permite o compartilhamento de dados entre comunidades de pesquisadores, viabilizando recursos e tempo, além de permitir uma significante interação entre pesquisadores de todo o mundo, principalmente brasileira, na troca de informações e compartilhamento de dados. Dados elementares relacionados às serpentes foram armazenados no banco de dados, bem como as atividades tóxicas, farmacológicas e enzimáticas dos componentes dos venenos, e ainda, as aplicações biotecnológicas dos produtos que podem ser obtidos destes venenos, abrangendo ainda dados clínicos e valores estatísticos dos acidentes ofídicos. Aspectos bioquímicos dos ensaios realizados em laboratório permitiram a construção de uma ferramenta para análise comparativa de estruturas primárias de PLA2s, depositadas em bancos de dados internacionais. Além da interatividade entre pesquisadores, em Fóruns de Discussões, o sistema conta com listas dos principais artigos publicados em periódicos indexados, e devidamente atualizados periodicamente, com revisões bibliográficas.Technological advances have been contributing, more and more, with biological and scientific areas, offering computational tools and specific systems which in silico data analysis supply reliable and fast results. The present project considers the development of an Internet portal for installation and use of laboratory data base for the main Brazilian serpents, with its respective venom and natural anti-venom, and the analysis of obtained data in pharmacological assays and biochemists. Using the easiest way of communication and interaction, the Internet allows sharing of data and information between communities of researchers around the world, especially for Brazilian researchers, making resources and time possible. Elementary data about serpents have been stored in the data base, as well as toxic, pharmacological and enzymatic activities of venom components, besides biotechnological applications of the products that can be obtained from these venom, enclosing clinical data and statistical values of ophidian accidents. Biochemists aspects of the assays carried through in laboratory allowed the construction of a comparative analysis tool for primary structures of PLA2s, deposited in international data bases. Beyond interactivity between researchers, in discussion forums, the system counts with lists of main articles published in indexed periodic, duly and constantly updated with bibliographical revisions.
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Hirtzlin-Pinçon, Olivier. "L'influence de la situation géopolitique au Moyen-Orient sur la génération des accords israélo-arabes depuis "Camp David I" : la frontière d'Israël." Phd thesis, Université des Sciences Sociales - Toulouse I, 2008. http://tel.archives-ouvertes.fr/tel-00300769.
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La question moyenne-orientale est dans l'actualité depuis 1948. C'est en cette année que se crée l'Etat d'Israël sur les décombres du mandat britannique en Palestine. Dès le commencement, la guerre va commencer à fixer les frontières entre Israël et ses voisins arabes. Cependant, après 1967, une nouvelle question va apparaître, celle des relations avec les Territoires occupés. En conséquence, l'Etat d'Israël aura deux questions frontalières à gérer : la question interétatique classique et la question interne avec les Palestiniens. Cette recherche tente de démontrer les voies employées par les différents acteurs régionaux et internationaux pour trouver une solution à cette question juridique qui cause l'instabilité régionale. On s'appuiera sur le droit, l'Histoire, la science politique (en particulier, l'étude des idéologies sioniste et arabiste) et les relations internationales pour trouver une cohérence aux réussites et aux échecs qui ont émaillé l'histoire du Moyen-Orient depuis 1948 et le fait qu'Israël n'ait encore que deux frontières internationalement reconnues, une avec l'Egypte et l'autre avec le royaume de Jordanie.
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Trotman, Jackson B. "New Insights into the Biochemistry and Cell Biology of RNA Recapping." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523896565730483.
Full textBooks on the topic "CAMP assay"
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Rossi, Maddalena, and Claudio Saragosa, eds. I territori della contemporaneità. Florence: Firenze University Press, 2019. http://dx.doi.org/10.36253/978-88-6453-805-1.
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Il testo raccoglie la rielaborazione di alcune delle tesi di laurea più significative prodotte, dal 2011 al 2015, nei corsi di laurea triennale in Pianificazione della città, del territorio e del paesaggio e di laurea magistrale in Pianificazione e progettazione della città e del territorio dell’Università di Firenze, con sede a Empoli. Le tesi trattano un panorama attuale e variegato di problematiche interne alla disciplina urbanistica, utilizzando metodologie, chiavi di lettura e prospettive di analisi assai diverse. Il territorio che emerge come protagonista delle narrazioni dei giovani autori è un oggetto complesso e pluristratificato, fatto di cose e relazioni, adagiato sui tempi lunghi della storia, teso sul presente e proiettato nel futuro, che continua a sollecitare loro domande, dubbi, curiosità e anche alcune fruttuose risposte. In sintesi, più che campi di discussione di un sapere acquisito e valido una volta per tutte, i lavori qui presentati rappresentano campi di riflessione e di sperimentazione, occasioni di costruzione incrementale di soluzioni creative da parte degli autori.
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Smith, Kenda. Notebook: Camper Queen Classy Sassy Smart Assy Camp Flamingo Women Notebook 6x9 Inch by Kenda Smith. Independently Published, 2021.
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Misra, Udayon. The Critical Forties I. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780199478361.003.0002.
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In the 1940s, the issues of immigration, land, and identity gained an urgency that had never been witnessed before. Under the different ministries led by Syed Muhammad Saadulla, immigration of Muslim peasants from East Bengal received a new impetus from the 1930s onwards, and the issue of land became a contentious one. Following the All India Muslim League’s Lahore Resolution of 1940, the issue of immigration acquired grave political overtones and became inextricably linked with the question of land and the identity of the indigenous Assamese and tribal populations. The details from the Assam Legislative Assembly debates reveal the diametrically opposite positions held by the Indian National Congress and the Muslim League on immigration, land, and identity. During this time the question of identity came to occupy a central place, and an attempt to do away with administrative measures such as the Line System created a highly explosive situation in the state.
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Lima, Tatiane do Nascimento, and Rogério Rodrigues Faria. Ecótono Cerrado Pantanal: meio ambiente e história natural. Editora Amplla, 2021. http://dx.doi.org/10.51859/amplla.ecp672.1121-0.
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O termo Bioma refere-se a uma área do espaço geográfico representada por um tipo uniforme de ambiente, dentro do qual é possível identificar características similares de macroclima, fitofisionomia, solo e altitude (WALTER, 1986). Dentro dessas áreas espécies surgiram e se desenvolveram em resposta à essas características do ambiente. Tal processo permite que por exemplo, dentro dessas áreas os vegetais apresentem aspectos, formas e processos fisiológicos característicos (CRAWLEY, 1989). Dessa maneira, a manutenção desses biomas, com suas características ambientais únicas, é de fundamental importância para a manutenção da biodiversidade e dos serviços ecossistêmicos que ali ocorrem (regulação climática, ciclo de matéria, segurança alimentar, entre outros) (PBMC/BPBES, 2018; JOLY et al., 2019). O Brasil é formado por seis grandes biomas: Amazônia, Caatinga, Cerrado, Mata Atlântica, Pampas e Pantanal (IBGE, 2019). Dentro desses ambientes são encontrados uma grande diversidade de fauna e flora e características únicas de relevo e clima. Essa variedade de biomas está relacionada a grande extensão territorial do Brasil e a sua posição geográfica. Todas essas características fazem do Brasil o maior detentor de biota continental do mundo, sendo estimado um valor entre 15% e 20% das aproximadamente 1,5 milhões de espécies descritas no planeta. Só de plantas vasculares os números mais recentes citados são de 56108 espécies, com 12400 (22%) endêmicas. Esses dados representam aproximadamente 22% do total mundial (LEWINSOHN; PRADO, 2002; SHEPHERD, 2002; HUBBELL, 2008; GIAM et al., 2010). Dentro desse contexto, os biomas Cerrado e Pantanal se integram por meio dos rios que nascem nos planaltos do Cerrado. Esses rios contribuem na formação do Pantanal, nas planícies inundáveis da bacia do Paraguai (BRASIL, 2007). No Domínio Cerrado, a dinâmica ambiental é proveniente de uma marcada sazonalidade climática com duas estações bem definidas, o período seco e o período chuvoso (ASSAD, 1994; SILVA, 2011). Essa sazonalidade climática modifica constantemente as propriedades do solo, da flora e da paisagem e a reestruturação de muitas comunidades (AMARAL et al., 2013; MALHEIROS, 2016). No Pantanal as áreas conhecidas como planícies de inundação se caracterizam pela presença de hábitats que variam de aquáticos a terrestres, de acordo com o grau de comunicação com o rio principal (PAZ; TUCCI, 2010). Os ciclos de secas e cheias são um importante fenômeno hídrico para a região, criando um sistema complexo e dinâmico (JUNK; DA SILVA, 1999; RESENDE, 2008). O Cerrado é uma das 25 áreas do mundo consideradas críticas para a conservação, devido à riqueza biológica e à alta pressão antrópica a que vem sendo submetido (MYERS et al., 2000). O Pantanal, por sua vez, é reconhecido mundialmente pela abundância de sua fauna (MITTERMEIER et al., 1990; HARRIS et al., 2005) e é considerado Reserva da Biosfera e Patrimônio Natural da Humanidade pela Unesco (BRASIL, 2018). O conhecimento dos aspectos que envolvem a fauna, a flora e as características dessas paisagens são de extrema importância para a sua conservação e preservação. As áreas de transição entre esses dois biomas, chamadas áreas de ecótono, se fazem presentes no estado do Mato Grosso do Sul. Nessa região, os biomas Cerrado e Pantanal possuem correlações quanto aos aspectos geomorfológicos e fitogeográficos (RODRIGUES et al., 2017). Na região o encontro entre o Planalto de Maracaju-Campo Grande e a Planície Pantaneira é uma área comum de elementos bióticos e abióticos entre o planalto e a planície (FILHO et al., 2009). A transição entre dois ecossistemas implica a existência de uma área com valores intermediários para diversos parâmetros ambientais (NEIFF, 2003). Por um lado, a área de transição pode gerar um aumento na biodiversidade, dado o fato dessas áreas apresentarem representantes de fauna e flora dos dois ecossistemas (VELOSO et al., 1991). Contudo, essas áreas de transição podem também representarem barreira ou área de isolamento com ecossistemas vizinhos (MALANSON, 1997). Desta forma, uma análise voltada para as áreas de ecótono entre esses dois biomas faz-se necessária, uma vez que a preservação de um depende da preservação do outro. Sobretudo para o entendimento de que essas paisagens de ecótono podem ser responsáveis pelo isolamento e amortecimento das alterações dentro dos biomas Cerrado e Pantanal. Este E-book traz estudos desenvolvidos na área de ecótono Cerrado Pantanal no município de Aquidauana (MS) e entorno. O município está localizado a 130 Km a oeste da capital Campo Grande. Aquidauana por se tratar de um município com influência dos biomas Cerrado e Pantanal, abriga uma grande biodiversidade, sendo citada pelo Ministério do Meio Ambiente (BRASIL, 2002) como área prioritária para conservação da biodiversidade. Na mesma via, o município se destaca por sua vocação turística e agropecuária, o que demanda atenção, devido ao processo de intensa ocupação e exploração antrópica dos recursos naturais. Dessa maneira, o conhecimento de suas características ambientais e dos processos ecológicos desempenhados por sua fauna e flora contribuem para sua preservação e manutenção.
Book chapters on the topic "CAMP assay"
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Peng, Grace E., and Mark von Zastrow. "A Live-Cell Imaging Assay for Nuclear Entry of cAMP-Dependent Protein Kinase Catalytic Subunits Stimulated by Endogenous GPCR Activation." In cAMP Signaling, 339–49. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2245-2_21.
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Cecon, Erika, Jean-Luc Guillaume, and Ralf Jockers. "Functional Investigation of Melatonin Receptor Activation by Homogenous cAMP Assay." In Melatonin, 179–88. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2593-4_22.
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Mazina, Olga, Anni Allikalt, Annika Heinloo, Reet Reinart-Okugbeni, Sergei Kopanchuk, and Ago Rinken. "cAMP Assay for GPCR Ligand Characterization: Application of BacMam Expression System." In Methods in Molecular Biology, 65–77. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2336-6_5.
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Lavogina, Darja, Tõnis Laasfeld, Maris-Johanna Tahk, Olga Kukk, Anni Allikalt, Sergei Kopanchuk, and Ago Rinken. "cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation." In Methods in Molecular Biology, 179–92. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1221-7_12.
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Bradley, Joe, and David McLoughlin. "Use of the DiscoveRx Hithunter cAMPII Assay for Direct Measurement of cAMP in Gs and Gi GPCRs." In Methods in Molecular Biology, 171–79. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-317-6_12.
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Maity, Chiranjit, Dipankar Ghosh, and Sonia Guha. "Assays for Intracellular Cyclic Adenosine Monophosphate (cAMP) and Lysosomal Acidification." In Methods in Molecular Biology, 161–78. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9488-5_14.
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Bassoni, Daniel L., Qumber Jafri, Sunitha Sastry, Mahesh Mathrubutham, and Tom S. Wehrman. "Characterization of G-Protein Coupled Receptor Modulators Using Homogeneous cAMP Assays." In Receptor Binding Techniques, 171–80. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-909-9_8.
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Walker, Madison F., and Benjamin R. Myers. "Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors." In Hedgehog Signaling, 175–84. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1701-4_15.
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"The Evolution of cAMP Assays." In Handbook of Assay Development in Drug Discovery, 261–86. CRC Press, 2006. http://dx.doi.org/10.1201/9781420015706-24.
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Kasila, Patricia, and Harry Harney. "The Evolution of cAMP Assays." In Handbook of Assay Development in Drug Discovery, 243–68. CRC Press, 2006. http://dx.doi.org/10.1201/9781420015706.ch19.
Full textConference papers on the topic "CAMP assay"
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Worzella, Tracy J., Tim Allison, Pete Stecha, Mei Cong, Chad Zimprich, Kevin Kershner, and Richard L. Somberg. "Abstract 3883: Miniaturizing the GloSensor™ cAMP assay for MC4R screening using the Echo® acoustic liquid handler." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-3883.
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Suga, K., Y. Uemura, T. Tsuijinaka, M. Sakon, J. Kambayashi, and T. Mori. "PROPERTIES OF PHOSPHATIDYLINOSITOL KINASE IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643807.
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We have reported the specific 32P-labelling in phosphatidyl-inositol-4-monophosphate(PIP) of intact platelets upon addition of the agents which elevate intracellular cAMP (Thrombos.Res.44, 155,1986).This event may be catalyzed by the action of Pl-kinase, the properties of which has not been elucidatedyet.Thereby, attempts were made to assay and to characterize PI-kinase of human platelets.Fresh lysed platelets prelabelled with 32P in cold Tris-HCl buffer containing 2mM EGTA were incubated at 37 C in the presence of MgCl2 for designated times and the phospholipids were extracted and analyzed by thin layer chromatography.32P-labelling in PIP was gradually increased in consort with the decreased labelling in PI-4,5-bisphosphate.As the changes in the labelling was not affected by the presence of apyrase and as the radioactive inositol trisphosphate was not detected,it was suggested that the changes is due to the action of phoshomono-esterase rather than PI-kinase or phospholipase C.When 32P-ATP was added to non-labelled lysed platelets upon incubation, 32P was labelled only into PIP and the amount was markedly increased until 5min. after incubation.Since the labelling was strongly inhibited by apyrase,it likely reflects the activity of Pl-kinase. The activity of PI-kinase thus measured required Mg2+ strictly for the activity and the maximal activity was obtained in the presence of 30mM Mg2+ .In contrast,it was markedly inhibited in the presence of Ca2+ (as low as 2mM Ca2+ in the presence of 2mM EGTA),which was compatible.with our previous findings with intact platelets. The activity of A-kinase was not inhibited by a low concentration of Ca2+ .Furthermore,the activity was inhibited by cAMP or dbcAMP in a dose related manner and no enhancement of the activity Was obtained by the addition of catalytic subunit of A-kinase,though a significant reduction in the activity was observed in the presence of inhibitor protein to A-kinase. From these observations,the following conclusions were obtained; l)The activity of Pl-kinase in lysed platelets may be determined by pulse labelling with 32P-ATP. 2)It requires Mg2+ absolutely and is inhibited by a very low concentration of Ca2+. 3)Pl-kinase is activated by A-kinase but the activated enzyme is inhibited by cAMP, suggesting the presence of feedback mechanism.
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SCHLEGEL, N., J. MOAKE, C. LOIRAT, M. F. HURTAUD, S. LEVY-TOLEDANO, and H. MATHIEU. "CHILDHOOD HEMOLYTIC UREMIC SYNDROME (HUS) : VON WILLEBRAND FACTOR (vWF) AND PLATELET AGGREGATING ACTIVITY (PAA) STUDIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643475.
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It has been suggested that a vWF High Molecular Weight Multi-mers (HMWM) decrease or a PAA were involved in the pathogenesis of HUS. We have studied 8 children (6 girls,_2 boys; 7 months-8_1/2 years old) with HUS : plasma creatinine /μmol/l; mean(range)/=306 (105-524), hemoglobin (g/100ml)-7(6.3-7.8), schistocytes (%)=8(1-18), platelets (x103/mm3)-57(10-115). The vWF was studied quantitatively (antigen ; vWF RAg assay) and qualitatively (multimeric pattern : immunoblotting and autoradiography). PAA studied by incubating the patient's platelet poor plasma (RPR) with washed normal platelets (aggregometer, % light transmission) and confirmed by Thromboxane B2 (TXB2) assay and [14C] Serotonine release study. The PAA was characterized by studying the in vitro effect of several platelet aggregation inhibitors, Immunoglobulins (Igs) and Fresh Frozen Plasma (FFP) on the platelet aggregation.An increase of vWF RAg (%) was observed in 6 cases : mean:330, and possibly related with renal failure. A vWF HMWM decrease was found in 3 patients : 2/3 with associated infection(E.Coli, Pneumococcus), 1/3 with severe hemolysis. Two of these 3 patients had a favourable renal outcome and 1 a severe course (chronic hemodialysis, Arterial Thrombotic MicroAngiopathy at renal histology).An important PAA was evidenced only in 1 patient : post bone-marrow graft HUS during neuroblastoma(NB),arterial hypertension and chronic renal failure. This PAA was Ca++, TXB2 and cAMP dependent; it was moderately inhibited in vitro by Igs and FFP, but persisted after 5 days of Igs infusion (0.3g/Kg/day). Treatment with aspirin and dipyridamole (10mg/Kg/day each) suppressed the patient platelet auto-aggregation although the PAA persisted (follow up:10months). The PAA did not seem to be related with the NB (absence of GD2 ganglioside, specific marker of NB); it could be related with anti platelet antibodies. The coexistence of the two abnormalities could not be demonstrated in our patients.In conclusion, a vWF HMWM decrease was found in 3 out of 8 children patients with HUS. Its presence was not correlated with the severity of the disease. We could demonstrate the presence of PAA during childhood HUS in only 1 post bone-marrow graft case. The PAA characterization is useful for therapeutic decisions and contributes to a better pathogenetic understanding.
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Barrett, Victoria J., Amanda Emmons, Alison J. Ford, and Richard Knowles. "In Vitro Pharmacological Characterisation Of GW642444, A Novel Long Acting ²2-agonist (LABA) Using Human Recombinant ²1/2/3 Adrenoceptor CAMP Assays." In American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a4451.
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Orendain, Adam, Jose Carrasco, Eniko T. Enikov, and Gholam Peyman. "Evaluation of Electro-Spun Tubular Scaffolds to Create an Anastomosis Using the CAM Assay." In ASME 2013 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/imece2013-64687.
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Central retinal vein occlusion (CRVO) is a vascular disease characterized by thrombosis of the retinal veins that can eventually lead to ischemia. Ischemic CRVO can then cause macular degeneration and neovascular glaucoma causing partial to full blindness. In this study, we determined the feasibility of electrospinning tubular scaffolds for treating CRVO and vascular disease. Electrospinning was utilized to produce customizable scaffolds from nano-bers using collagen type I. Scaffolds were treated with glutaraldehyde, glycine, ethanol, UV light, and combinations of the treatments for the purpose cross-linking and to study their angiogenic effects. Structural properties of the scaffolds were analyzed with scanning electron micrsoscopy (SEM). Scaffolds were immobilized with human recombinant vascular endothelial growth factor (rhVEGF165) to investigate the drug-delivering abilities of the electrospun materials and as a method to produce vascularization. The chick chorioallantoic membrane (CAM) assay was used to examine the effects of VEGF immobilizations and to evaluate the feasibility of creating an anastomosis to treat CRVO. Collagen onplants (non-electrospun) and electrospun implants were made on day 10 of embryonic development. Findings show collagen loaded with rhVEGF165 had improved vasculature and pro-angiogenic properties. The present study suggests that collagen can immobilize and release growth factor, be electrospun to mimic the ultrastructure of native blood vessels, and holds promise for vascular tissue engineering.
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Jee, Kwang Yong, Hong Joo Ahn, Se Chul Sohn, Su Ho Han, and Ki Seop Choi. "Derivation of the Korean Radwaste Scaling Factor." In The 11th International Conference on Environmental Remediation and Radioactive Waste Management. ASMEDC, 2007. http://dx.doi.org/10.1115/icem2007-7297.
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The concentrations of several radionuclides in low and intermediate level radioactive waste (LILW) drums have to be determined before shipping to disposal facilities. A notice, by the Ministry of Science and Technology (MOST) of the Korean Government, related to the disposal of LILW drums came into effect at the beginning of 2005, with regards to a radionuclide regulation inside a waste drum. MOST allows for an indirect radionuclide assay using a scaling factor to measure the inventories due to the difficulty of nondestructively measuring the essential α and β-emitting nuclides inside a drum. That is, a scaling factor calculated through a correlation of the α or β-emitting nuclide (DTM, Difficult-To-Measure) with a γ-emitting nuclide (ETM, Easy-To-Measure) which has systematically similar properties with DTM nuclides. In this study, radioactive wastes, such as spent resin and dry active waste which were generated at different sites of a PWR and a site of a PHWR type Korean NPP, were partially sampled and analyzed for regulated radionuclides by using radiochemical methods. According to a reactor type and a waste form, the analysis results of each radionuclide were classified. Korean radwaste scaling factor was derived from database of radionuclide concentrations.
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Ahmad, Salma, Hanan Nazar, Nouralhuda Alatieh, Maryam Al-Mansoob, Zainab Farooq, Muna Yusuf, and Allal Ouhtit. "Validation of Novel Transcriptional Targets that Underpin CD44-promoted breast cancer cell invasion." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2021. http://dx.doi.org/10.29117/quarfe.2021.0153.
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Introduction: Breast cancer (BC) is the most common cancer worldwide, and metastasis is its worst aspect and the first cause of death. Metastasis is a multistep process, where an invasion is a recurring event. The process of BC cell invasion involves three major factors, including cell adhesion molecules (CAM), proteinases and Growth factors.CD44, a family of CAM proteins and the hyaluronic acid (HA) cell surface receptor, acts as cell differentiation, cell migration/invasion and apoptosis regulator. Rationale: We have previously established a tetracycline (Tet)-OFF-regulated expression system, both in vitro and in vivo (Hill et al, 2006). As a complementary approach, the highly metastatic MDA-MB-231 BC cells expressing high levels of endogenous CD44s (the standard form of CD44), was cultured in the presence and absence of 50 µg/ml of HA. RNA samples were isolated from both cell experimental models, and microarray analysis (12K CHIP from Affymetrix) was applied. More than 200 CD44s transcriptional target genes were identified and were sub-divided into groups of genes based on their function: cell motility, cytoskeletal organization, ability to degrade ECM, and cell survival. Hypothesis: Among these 200 identified genes, we selected seven genes (ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1, and SOD2) and hypothesized that based on evidence from literature, these genes are potential novel targets of CD44-downstream signaling mediating BC cell invasion. Specific Aims: Pursuant to this goal, we proposed the following objectives: 1- Structural validation of ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1 and SOD2 as novel transcriptional targets of CD44/HA-downstream signaling at both RNA and Protein level using reverse transcription polymerase chain reaction (RT-PCR) and Western Blot respectively. 2-Functional validation of ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1and SOD2 as novel transcriptional targets that underpin CD44-promoted BC cell migration using wound healing assay after the transfection with siRNA. Innovation/Consclusion: This study validated seven transcriptional targets of CD44/HA-downstream signaling promoting BC cell invasion. Ongoing experiments aim to dissect the signaling pathways that link CD44 activation by HA to the transcription of these seven genes.
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Lefrère, J. J., D. Gozin, J. P. Soulier, P. Mavier, L. Bettan, and D. Dhumeaux. "SPECIFICITY OF INCREASED DES-GAMMA-CARBOXYPROTHROMBIN AS A MARKER OF HEPATOCELLULAR CARCINOMA AFTER VITAMIN K INJECTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644319.
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An elevation of des-gamma-carboxyprothrombin (DCP) has been observed in about 70 % of cases of hepatocellular carcinoma (HCC). Howewer, an increased DCP is not specific of HCC. Oral anticoagulant therapy increases the DCP level by preventing the gamma-carboxylation of prothrombin : thereafter an increased DCP can not be used as an HCC marker before three weeks have elapsed after stopping anti vitamin K therapy. Furthermore, since vitamin K is necessary for the gamma-carboxylation of vitamin K dependent factors, a vitamin K deficiency increases the DCP level long before the modification of the prothrombin time. It is thus imperative to eliminate an underlying vitamin K deficiency before attributing an increased DCP to a HCC. We used a method of DCP assay using staphylocoagulase. We studied the effect of an intravenous injection of 20 mg of vitamin K1 on DCP level in 7 patients with histologically proven HCC and in 10 patients with various disorders (5 alcoholic cirrhosis, 1 chronic hepatitis, 4 pancreatic cancer). All these 17 patients had increased DCP before vitamin K injection. In a second sampling obtained 15 days or more after injection, only the 7 patients with HCC kept increased DCP level. In patients of both categories in whom we obtained intermediary samplings, we observed that the DCP level decreased In all cases. The normalisation of the DCP level was lasting only in those patients without HCC, confirming the hypothesis of an underlying vitamin K deficiency ; this decrease was very transitory in those patients with HCC, suggesting that the elevated DCP came from a yet unexplained (but not linked to a vitamin K deficiency) mechanism. We may conclude that an increased DCP level 15 days after vitamin K injection may constitute a specific marker of HCC.
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Mohamed, Islam, Ahmed Moahmed, Mennatallah Abdelkader, Alaaeldin Saleh, and Ala-Eddin Al-Moustafa. "Elaeagnus Angustifolia: a Promising Medicinal Plant for Cancer Theraby." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0124.
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Introduction: Elaeagnus angustifolia (EA) is a medicinal plant that has been used for centuries in treating many human diseases, in the Middle East, including fever, amoebic dysentery, gastrointestinal problems. However, the effect of EA plant extract on human cancer progression especially oral malignancy has not been investigated yet. Thus, first we examined the effect of EA flower extract on angiogenesis in ovo, and on selected parameters in human oral cancer cells. Materials and methods: Chorioallantoic membranes (CAMs) of chicken embryos at 3-7 days of incubation were used to assess the effect EAflower plant extract on angiogenesis. Meanwhile, cell proliferation, soft agar, cell cycle, cell invasion and cell wounding assays were performed to explore the outcome of EA plant extract on FaDu and SCC25 oral cancer cell lines. On the other hand, western blot analysis was carried out to evaluate E-cadherin and Erk1/Erk2 expression and activation, respectively, in FaDu and SCC25 under the effect of EA extract. Results: Our data show that EA extract inhibits cell proliferation and colony formation, in addition to the initiation of Scell cycle arrest and reductionof G1/G2 phases. In parallel, EA extract provokes differentiation to an epithelial phenotype “mesenchymal-epithelial transition: MET” which is the opposite of “epithelial-mesenchymal transition, EMT”: an important event in cell invasion and metastasis. Thus, EA extract causes a dramatic decrease in cell motility and invasion abilities of FaDu and SCC25 cancer cells in comparison with their controls. These changes are accompanied by an up-regulation of E-cadherin expression. The molecular pathway analysis of the EA flower extract reveals that it can inhibit the phosphorylation of Erk1/Erk2, which could be behind the inhibition of angiogenesis, the initiation of MET event and the overexpression of E-cadherin. Conclusions: Our findings indicate that EA plant extract can downgrade human oral cancer progression by the inhibition of angiogenesis and cell invasion via Erk1/Erk2 signaling pathways.
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Zambalde, Erika Pereira, Ana Carolina Rodrigues, Rubens Silveira Lima, Enilze Maria Souza Fonseca Ribeiro, and Jaqueline Carvalho Oliveira. "TLNC-UC.147, A NOVEL LONG RNA (lncRNA) FROM AN ULTRACONSERVED REGION AS POTENTIAL BIOMARKER IN LUMINAL A BREAST CANCER." In Scientifc papers of XXIII Brazilian Breast Congress - 2021. Mastology, 2021. http://dx.doi.org/10.29289/259453942021v31s1052.
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Introduction: Long RNAs are non-coding RNAs with more than 200 nucleotides in length, with essential regulatory roles in several biological processes, including in breast cancer (BC). The human genome contains 481 ultraconserved regions, which are genomic stretches of over 200 base pairs conserved among humans, rats, and mice. Most of these regions are transcriptionally active (T-UCRs), and several are differentially expressed in tumors. Some T-UCRs have been functionally characterized, but few have been associated with BC. Objectives: In this study, we aimed to expand the knowledge of T-UCRs in BC and characterize the lnc-uc.147, a long RNA transcribed from an ultraconserved region. Methods: We evaluated the expression level of 481 T-UCRs and their association with clinical parameters from TCGA data. For confirmation, 102 Brazilian BC samples were analyzed by RT-qPCR. Cytosolic and nuclear cell fractions and RT-qPCRs were done to determine the cell compartment of the transcript. Northern blotting and RACE were performed to determine the sequence and precise size of lnc-uc.147. Using two luminal cell lines (CAMA and BT474), a siRNA-based approach was applied to investigate the effects of lnc-uc.147 knockdown in cell viability, colony formation, and apoptosis level. To understand the interactions of lnc-uc.147 and proteins, we performed a pull-down assay. Results: Using TCGA (The Cancer Genome Atlas) data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with estrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. We found that uc.147 is highly expressed in luminal A and B patients, which was also confirmed in Brazilian samples. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic transcript. The silencing of uc.147 increases apoptosis, arrests the cell cycle and reduces cell viability and colony formation in luminal BC cell lines. Additionally, we identified 19 proteins that interact with uc.147 through mass spectrometry. These proteins are mainly involved in cytoskeletal and centrosome organization as well as in epithelial-mesenchymal transition. Conclusions: We show herein evidence that neoplastic BC cells exhibit a unique expression profile of T-UCRs. This study characterized the lnc-uc.147, a transcript that has never been described before. Indeed, lnc-uc.147 has an oncogenic effect in the luminal BC cell line and can interact with proteins. Furthermore, uc.147 has the potential as a BC prognostic marker in luminal patients.
Reports on the topic "CAMP assay"
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Lance, Richard, and Xin Guan. Variation in inhibitor effects on qPCR assays and implications for eDNA surveys. Engineer Research and Development Center (U.S.), August 2021. http://dx.doi.org/10.21079/11681/41740.
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Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.
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Altstein, Miriam, and Ronald J. Nachman. Rational Design of Insect Control Agent Prototypes Based on Pyrokinin/PBAN Neuropeptide Antagonists. United States Department of Agriculture, August 2013. http://dx.doi.org/10.32747/2013.7593398.bard.
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The general objective of this study was to develop rationally designed mimetic antagonists (and agonists) of the PK/PBAN Np class with enhanced bio-stability and bioavailability as prototypes for effective and environmentally friendly pest insect management agents. The PK/PBAN family is a multifunctional group of Nps that mediates key functions in insects (sex pheromone biosynthesis, cuticular melanization, myotropic activity, diapause and pupal development) and is, therefore, of high scientific and applied interest. The objectives of the current study were: (i) to identify an antagonist biophores (ii) to develop an arsenal of amphiphilic topically active PK/PBAN antagonists with an array of different time-release profiles based on the previously developed prototype analog; (iii) to develop rationally designed non-peptide SMLs based on the antagonist biophore determined in (i) and evaluate them in cloned receptor microplate binding assays and by pheromonotropic, melanotropic and pupariation in vivo assays. (iv) to clone PK/PBAN receptors (PK/PBAN-Rs) for further understanding of receptor-ligand interactions; (v) to develop microplate binding assays for screening the above SMLs. In the course of the granting period A series of amphiphilic PK/PBAN analogs based on a linear lead antagonist from the previous BARD grant was synthesized that incorporated a diverse array of hydrophobic groups (HR-Suc-A[dF]PRLa). Others were synthesized via the attachment of polyethylene glycol (PEG) polymers. A hydrophobic, biostablePK/PBAN/DH analog DH-2Abf-K prevented the onset of the protective state of diapause in H. zea pupae [EC50=7 pmol/larva] following injection into the preceding larval stage. It effectively induces the crop pest to commit a form of ‘ecological suicide’. Evaluation of a set of amphiphilic PK analogs with a diverse array of hydrophobic groups of the formula HR-Suc-FTPRLa led to the identification of analog T-63 (HR=Decyl) that increased the extent of diapause termination by a factor of 70% when applied topically to newly emerged pupae. Another biostablePK analog PK-Oic-1 featured anti-feedant and aphicidal properties that matched the potency of some commercial aphicides. Native PK showed no significant activity. The aphicidal effects were blocked by a new PEGylated PK antagonist analog PK-dF-PEG4, suggesting that the activity is mediated by a PK/PBAN receptor and therefore indicative of a novel and selective mode-of-action. Using a novel transPro mimetic motif (dihydroimidazole; ‘Jones’) developed in previous BARD-sponsored work, the first antagonist for the diapause hormone (DH), DH-Jo, was developed and shown to block over 50% of H. zea pupal diapause termination activity of native DH. This novel antagonist development strategy may be applicable to other invertebrate and vertebrate hormones that feature a transPro in the active core. The research identifies a critical component of the antagonist biophore for this PK/PBAN receptor subtype, i.e. a trans-oriented Pro. Additional work led to the molecular cloning and functional characterization of the DH receptor from H. zea, allowing for the discovery of three other DH antagonist analogs: Drosophila ETH, a β-AA analog, and a dF analog. The receptor experiments identified an agonist (DH-2Abf-dA) with a maximal response greater than native DH. ‘Deconvolution’ of a rationally-designed nonpeptide heterocyclic combinatorial library with a cyclic bis-guanidino (BG) scaffold led to discovery of several members that elicited activity in a pupariation acceleration assay, and one that also showed activity in an H. zea diapause termination assay, eliciting a maximal response of 90%. Molecular cloning and functional characterization of a CAP2b antidiuretic receptor from the kissing bug (R. prolixus) as well as the first CAP2b and PK receptors from a tick was also achieved. Notably, the PK/PBAN-like receptor from the cattle fever tick is unique among known PK/PBAN and CAP2b receptors in that it can interact with both ligand types, providing further evidence for an evolutionary relationship between these two NP families. In the course of the granting period we also managed to clone the PK/PBAN-R of H. peltigera, to express it and the S. littoralis-R Sf-9 cells and to evaluate their interaction with a variety of PK/PBAN ligands. In addition, three functional microplate assays in a HTS format have been developed: a cell-membrane competitive ligand binding assay; a Ca flux assay and a whole cell cAMP ELISA. The Ca flux assay has been used for receptor characterization due to its extremely high sensitivity. Computer homology studies were carried out to predict both receptor’s SAR and based on this analysis 8 mutants have been generated. The bioavailability of small linear antagonistic peptides has been evaluated and was found to be highly effective as sex pheromone biosynthesis inhibitors. The activity of 11 new amphiphilic analogs has also been evaluated. Unfortunately, due to a problem with the Heliothis moth colony we were unable to select those with pheromonotropic antagonistic activity and further check their bioavailability. Six peptides exhibited some melanotropic antagonistic activity but due to the low inhibitory effect the peptides were not further tested for bioavailability in S. littoralis larvae. Despite the fact that no new antagonistic peptides were discovered in the course of this granting period the results contribute to a better understanding of the interaction of the PK/PBAN family of Nps with their receptors, provided several HT assays for screening of libraries of various origin for presence of PK/PBAN-Ragonists and antagonists and provided important practical information for the further design of new, peptide-based insecticide prototypes aimed at the disruption of key neuroendocrine physiological functions in pest insects.
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Yaron, Zvi, Martin P. Schreibman, Abigail Elizur, and Yonathan Zohar. Advancing Puberty in the Black Carp (Mylopharyngodon Piceus) and the Striped Bass (Morone Saxatilis). United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568102.bard.
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The black carp (bc)GtH IIb cDNA was amplified and isolated, cloned and sequenced. Comparison of the bcGtH IIb deduced a.a. sequence with that of GtH IIb from other teleosts revealed high homology to cyprinid species and a lower homology to salmonid or perciform fish. The gene coding for the GtH IIb was isolated and sequenced. Three bc recombinant phages which hybridized to the goldfish GtH Ib cDNA probe were isolated and are currently being characterized. The region coding for the mature GtH IIb was expressed in a bacterial expression vector resulting in the production of a recombinant protein. In vitro folding resulted in a protein only 1.3% of which displaced the native common carp GtH II in a RIA. Therefore, the common carp GtH RIA was utilized for the physiological studies at the current phase of the project. Two non-functional sites were identified along the brain-pituitary gonadal axis in the immature black carp. The pituitary is refractory to GnRH stimulation due to a block proximal to the activation of PKA and PKC probably at the level of GnRH receptors. The gonads, although capable of producing steroids, are refractory to gonadotropic stimulation but do respond to cAMP antagonists, indicating a block at the GtH receptor level. Attempts to advance puberty in 2 and 3 y old black carp showed that testosterone (T) stimulates GtH synthesis in the pituitary and increases its sensitivity to GnRh. A 2 month treatment combining T+GnRH increased the circulating GFtH level in 3 y old fish. Addition of domperidone to such a treatment facilitated both the accumulation of GtH in the pituitary and its response to GnRH. The cDNA of striped bass GtH a, Ib and IIb subunits were amplified, isolated, cloned and sequenced, and their deduced a.a. sequences were compared with those of other teleosts. A ribonuclease protection assay was developed for a sensitive and simultaneous determination of all GtH subunits, and of b-actin mRNAs of the striped bass. GnRH stimulated dramatically the expression of the a and GtH IIb subunits but the level of GtH Ib mRNA increased only moderately. These findings suggest that GtH-II, considered in salmonids to be involved only in final stages of gametogenesis, can be induced by GnRH to a higher extent than GtH-I in juvenile striped bass. The native GtH II of the striped bass was isolated and purified, and an ELISA for its determination was developed. The production of all recombinant striped bass GtH subunits is in progress using the insect cell (Sf9) culture and the BAC-TO-BAC baculovirus expression system. A recombinant GtH IIb subunit has been produced already, and its similarity to the native subunit was confirmed. The yield of the recombinant glycoprotein can reach 3.5 mg/ml after 3 days culture. All male striped bass reach puberty after 3 y. However, precocious puberty was discovered in 1 and 2 y old males. Females become vitellogenic during their 4th year. In immature 2 y old females, T treatment elevates the pituitary GtH II content while GnRH only potentiates the effect. However, in males GnRH and not T affects GtH accumulation in the pituitary. Neither GnRH, nor T treatment resulted in gonadal growth in 2 y old striped bass, indicating that either the accumulated GtH II was not released, or if released, the gonads were refractory to GtH stimulation, similar to the situation in the immature black carp. In 3 y old female striped bass, 150 day GnRHa treatment resulted in an increase in GSI, while T treatment, with or without GnRHa, resulted in a decrease in oocyte diameter, similar to the effect seen in the black carp. Further attempts to advance puberty in both fish species should take into account the positive effect of T on pituitary GtH and its negative effect of ovarian growth.
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Bercovier, Herve, and Ronald P. Hedrick. Diagnostic, eco-epidemiology and control of KHV, a new viral pathogen of koi and common carp. United States Department of Agriculture, December 2007. http://dx.doi.org/10.32747/2007.7695593.bard.
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Original objectives and revisions-The proposed research included these original objectives: field validation of diagnostic tests (PCR), the development and evaluation of new sensitive tools (LC-PCR/TaqManPCR, antibody detection by ELISA) including their use to study the ecology and the epidemiology of KHV (virus distribution in the environment and native cyprinids) and the carrier status of fish exposed experimentally or naturally to KHV (sites of virus replication and potential persistence or latency). In the course of the study we completed the genome sequence of KHV and developed a DNA array to study the expression of KHV genes in different conditions. Background to the topics-Mass mortality of koi or common carp has been observed in Israel, USA, Europe and Asia. These outbreaks have reduced exports of koi from Israel and have created fear about production, import, and movements of koi and have raised concerns about potential impacts on native cyprinid populations in the U.S.A. Major conclusions-A suite of new diagnostic tools was developed that included 3 PCR assays for detection of KHV DNA in cell culture and fish tissues and an ELISA assay capable of detecting anti-KHV antibodies in the serum of koi and common carp. The TKPCR assay developed during the grant has become an internationally accepted gold standard for detection of viral DNA. Additionally, the ELISA developed for detecting serum anti-KHV antibodies is now in wide use as a major nonlethal screening tool for evaluating virus status of koi and common carp populations. Real time PCR assays have been able to detect viral DNA in the internal organs of survivors of natural and wild type vaccine exposures at 1 and 10³ genome equivalents at 7 months after exposure. In addition, vaccinated fish were able to transmit the virus to naive fish. Potential control utilizing hybrids of goldfish and common carp for production demonstrated they were considerably more resistant than pure common carp or koi to both KHV (CyHV-3). There was no evidence that goldfish or other tested endemic cyprinids species were susceptible to KHV. The complete genomic sequencing of 3 strains from Japan, the USA, and Israel revealed a 295 kbp genome containing a 22 kbp terminal direct repeat encoding clear gene homologs to other fish herpesviruses in the family Herpesviridae. The genome encodes156 unique protein-coding genes, eight of which are duplicated in the terminal repeat. Four to seven genes are fragmented and the loss of these genes may be associated with the high virulence of the virus. Viral gene expression was studies by a newly developed chip which has allowed verification of transcription of most all hypothetical genes (ORFs) as well as their kinetics. Implications, both scientific and agricultural- The results from this study have immediate application for the control and management of KHV. The proposal provides elements key to disease management with improved diagnostic tools. Studies on the ecology of the virus also provide insights into management of the virus at the farms that farmers will be able to apply immediately to reduce risks of infections. Lastly, critical issues that surround present procedures used to create “resistant fish” must be be resolved (e.g. carriers, risks, etc.). Currently stamping out may be effective in eradicating the disease. The emerging disease caused by KHV continues to spread. With the economic importance of koi and carp and the vast international movements of koi for the hobby, this disease has the potential for even further spread. The results from our studies form a critical component of a comprehensive program to curtail this emerging pathogen at the local, regional and international levels.
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Hedrick, Ronald, and Herve Bercovier. Characterization and Control of KHV, A New Herpes Viral Pathogen of Koi and Common Carp. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695871.bard.
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In this project we proposed to characterize the virus genome and the structural virion polypeptides to allow development of improved diagnostic approaches and potential vaccination strategies. These goals have been mostly achieved and the corresponding data were published in three papers (see below) and three more manuscripts are in preparation. The virion polypeptides of KHV strains isolated from USA (KHV-U) and Israel (KHV-I) were found to be identical. Purified viral DNA analyzed with a total of 5 restriction enzymes demonstrated no fragment length polymorphism between KHV-I and KHV-U but both KHV isolates differed significantly from the cyprinid herpesvirus (CHV) and the ictalurid herpesvirus (channel catfish virus or CCV). Using newly obtained viral DNA sequences two different PCR assays were developed that need to be now further tested in the field. We determined by pulse field analysis that the size of KHV genome is around 280 kbp (1-1. Bercovier, unpublished results). Sequencing of the viral genome of KHV has reached the stage where 180 kbp are sequenced (twice and both strands). Four hypothetical genes were detected when DNA sequences were translated into amino acid sequences. The finding of a gene of real importance, the thymidine kinase (TK) led us to extend the study of this specific gene. Four other genes related to DNA synthesis were found. PCR assays based on defined sequences were developed. The PCR assay based on TK gene sequence has shown improved sensitivity in the detection of KHV DNA compared to regular PCR assays. </P> <P><SPAN>With the ability to induce experimental infections in koi with KHV under controlled laboratory conditions we have studied the progress and distribution of virus in host tissues, the development of immunity and the establishment of latent infections. Also, we have investigated the important role of water temperature on severity of infections and mortality of koi following infections with KHV. These initial studies need to be followed by an increased focus on long-term fate of the virus in survivors. This is essential in light of the current "controlled exposure program" used by farmers to produce KHV "naturally resistant fish" that may result in virus or DNA carriers. </SPAN></P> <P><SPAN>The information gained from the research of this project was designed to allow implementation of control measures to prevent the spread of the virus both by improved diagnostic approaches and preventive measures. We have accomplished most of these goals but further studies are needed to establish even more reliable methods of prevention with increased emphases on improved diagnosis and a better understanding of the ecology of KHV. </SPAN>
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Elroy-Stein, Orna, and Dmitry Belostotsky. Mechanism of Internal Initiation of Translation in Plants. United States Department of Agriculture, December 2010. http://dx.doi.org/10.32747/2010.7696518.bard.
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Original objectives Elucidation of PABP's role in crTMV148 IRES function in-vitro using wheat germ extract and krebs-2 cells extract. Fully achieved. Elucidation of PABP's role in crTMV148 IRES function in-vivo in Arabidopsis. Characterization of the physical interactions of PABP and other potential ITAFs with crTMV148 IRES. Partly achieved. To conduct search for additional ITAFs using different approaches and evaluate the candidates. Partly achieved. Background of the topic The power of internal translation via the activity of internal ribosomal entry site (IRES) elements allow coordinated synthesis of multiple gene products from a single transcription unit, and thereby enables to bypass the need for sequential transformation with multiple independent transgenes. The key goal of this project was to identify and analyze the IRES-trans-acting factors (ITAFs) that mediate the activity of a crucifer-infecting tobamovirus (crTMV148) IRES. The remarkable conservation of the IRES activity across the phylogenetic spectrum (yeast, plants and animals) strongly suggests that key ITAFs that mediate its activity are themselves highly conserved. Thus, crTMV148 IRES offers opportunity for elucidation of the fundamental mechanisms underlying internal translation in higher plants in order to enable its rational manipulation for the purpose of agricultural biotechnology. Major conclusions and achievements. - CrTMV IRES requires PABP for maximal activity. This conclusion was achieved by PABP depletion and reconstitution of wheat germ- and Krebs2-derived in-vitro translation assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p. - Mutations in the internal polypurine tract of the IRES decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays. - Mutations in the internal polypurine tract decrease IRES activity in-vivo. - The 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap. - In-vivo assembled RNPs containing proteins specifically associated with the IRES were purified from HEK293 cells using the RNA Affinity in Tandem (RAT) approach followed by their identification by mass spectroscopy. - This study yielded a list of potential protein candidates that may serve as ITAFs of crTMV148 IRES activity, among them are a/b tubulin, a/g actin, GAPDH, enolase 1, ribonuclease/angiogenin inhibitor 1, 26S proteasome subunit p45, rpSA, eEF1Bδ, and proteasome b5 subunit. Implications, both scientific and agriculture. The fact that the 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the IRES sequence and the 3'-poly(A). This has an important scientific implication related to IRES function in general.
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Levy, Maggie, Raymond Zielinski, and Anireddy S. Reddy. IQD1 Function in Defense Responses. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7699842.bard.
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The main objective of the proposed research was to study IQD1's mechanism of action and elucidate its role in plant protection. Preliminary experiments suggest that IQD1 binds CaM in a Ca²⁺-dependent manner and functions in general defense responses. We propose to identify proteins and genes that interact with IQD1, which may provide some clues to its mechanism of action. We also plan to dissect IQD1's integration in defense pathways and to study and modulate its binding affinity to CaM in order to enhance crop resistance. Our specific objectives were: (1) Analysis of IQD1's CaM-binding properties; (2) Identification of IQD1 targets;(3) Dissection of IQD1 integration into defense signaling pathways. Analysis of IQD1's CaM-binding properties defined four potential classes of sequences that should affect CaM binding: one is predicted to raise the affinity for Ca²⁺-dependent interaction but have no effect on Ca²⁺-independent binding; a second is predicted to act like the first mutation but eliminate Ca²⁺-independent binding; a third has no predicted effect on Ca²⁺-dependent binding but eliminates Ca²⁺-independent binding; and the fourth is predicted to eliminate or greatly reduce both Ca²⁺-dependent and Ca²⁺-independent binding. Following yeast two hybrid analysis we found that IQD1 interact with AtSR1 (Arabidopsis thalianaSIGNALRESPONSIVE1), a calcium/calmodulin-binding transcription factor, which has been shown to play an important role in biotic and abiotic stresses. We tested IQD1 interaction with both N-terminal or C-terminal half of SR1. These studies have uncovered that only the N-terminal half of the SR1 interacts with the IQD1. Since IQD1 has an important role in herbivory, its interaction with SR1 suggests that it might also be involved in plant responses to insect herbivory. Since AtSR1, like IQD1, is a calmodulin-binding protein and the mutant showed increased sensitivity to a herbivore, we analyzed WT, Atsr1 and the complemented line for the levels of GS to determine if the increased susceptibility of Atsr1 plants to T. ni feeding is associated with altered GS content. In general, Atsr1 showed a significant reduction in both aliphatic and aromatic GS levels as compared to WT. In order to study IQD1's molecular basis integration into hormone-signaling pathways we tested the epistatic relationships between IQD1 and hormone-signaling mutants. For that purpose we construct double mutants between IQD1ᴼXᴾ and mutants defective in plant-hormone signaling and GS accumulation. Epitasis with SA mutant NahG and npr1-1 and JA mutant jar1-1 suggested IQD1 function is dependent on both JA and SA as indicated by B. cinerea infection assays. We also verified the glucosinolate content in the crosses siblings and found that aliphatic GSL content is reduced in the double transgenic plants NahG:IQD1ᴼXᴾ as compare to parental lines while the aliphatic GSL content in the npr1-1:IQD1ᴼXᴾ and jar1-1: IQD1ᴼXᴾ double mutants was intimidated to the parental lines. This suggests that GSL content dependency on SA is downstream to IQD1. As a whole, this project should contribute to the development of new defense strategies that will improve crop protection and reduce yield losses and the amount of pesticides required; these will genuinely benefit farmers, consumers and the environment.
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Altstein, Miriam, and Ronald Nachman. Rationally designed insect neuropeptide agonists and antagonists: application for the characterization of the pyrokinin/Pban mechanisms of action in insects. United States Department of Agriculture, October 2006. http://dx.doi.org/10.32747/2006.7587235.bard.
Full textAbstract:
The general objective of this BARD project focused on rationally designed insect neuropeptide (NP) agonists and antagonists, their application for the characterization of the mechanisms of action of the pyrokinin/PBAN (PK-PBAN) family and the development of biostable, bioavailable versions that can provide the basis for development of novel, environmentally-friendly pest insect control agents. The specific objectives of the study, as originally proposed, were to: (i) Test stimulatory potencies of rationally designed backbone cyclic (BBC) peptides on pheromonotropic, melanotropic, myotropic and pupariation activities; (ii) Test the inhibitory potencies of the BBC compounds on the above activities evoked either by synthetic peptides (PBAN, LPK, myotropin and pheromonotropin) or by the natural endogenous mechanism; (iii) Determine the bioavailability of the most potent BBC compounds that will be found in (ii); (iv) Design, synthesize and examine novel PK/PBAN analogs with enhanced bioavailability and receptor binding; (v) Design and synthesize ‘magic bullet’ analogs and examine their ability to selectively kill cells expressing the PK/PBAN receptor. To achieve these goals the agonistic and antagonistic activities/properties of rationally designed linear and BBC neuropeptide (NP) were thoroughly studied and the information obtained was further used for the design and synthesis of improved compounds toward the design of an insecticide prototype. The study revealed important information on the structure activity relationship (SAR) of agonistic/antagonistic peptides, including definitive identification of the orientation of the Pro residue as trans for agonist activity in 4 PK/PBANbioassays (pheromonotropic, pupariation, melanotropic, & hindgut contractile) and a PK-related CAP₂b bioassay (diuretic); indications that led to the identification of a novel scaffold to develop biostbiostable, bioavailable peptidomimetic PK/PBANagonists/antagonists. The work led to the development of an arsenal of PK/PBAN antagonists with a variety of selectivity profiles; whether between different PKbioassays, or within the same bioassay between different natural elicitors. Examples include selective and non-selective BBC and novel amphiphilic PK pheromonotropic and melanotropic antagonists some of which are capable of penetrating the moth cuticle in efficacious quantities. One of the latter analog group demonstrated unprecedented versatility in its ability to antagonize a broad spectrum of pheromonotropic elicitors. A novel, transPro mimetic motif was proposed & used to develop a strong, selective PK agonist of the melanotropic bioassay in moths. The first antagonist (pure) of PK-related CAP₂b diuresis in flies was developed using a cisPro mimetic motif; an indication that while a transPro orientation is associated with receptor agonism, a cisPro orientation is linked with an antagonist interaction. A novel, biostablePK analog, incorporating β-amino acids at key peptidase-susceptible sites, exhibited in vivo pheromonotropic activity that by far exceeded that of PBAN when applied topically. Direct analysis of neural tissue by state-of-the-art MALDI-TOF/TOF mass spectrometry was used to identify specific PK/PK-related peptides native to eight arthropod pest species [house (M. domestica), stable (S. calcitrans), horn (H. irritans) & flesh (N. bullata) flies; Southern cattle fever tick (B. microplus), European tick (I. ricinus), yellow fever mosquito (A. aegypti), & Southern Green Stink Bug (N. viridula)]; including the unprecedented identification of mass-identical Leu/Ile residues and the first identification of NPs from a tick or the CNS of Hemiptera. Evidence was obtained for the selection of Neb-PK-2 as the primary pupariation factor of the flesh fly (N. bullata) among native PK/PK-related candidates. The peptidomic techniques were also used to map the location of PK/PK-related NP in the nervous system of the model fly D. melanogaster. Knowledge of specific PK sequences can aid in the future design of species specific (or non-specific) NP agonists/antagonists. In addition, the study led to the first cloning of a PK/PBAN receptor from insect larvae (S. littoralis), providing the basis for SAR analysis for the future design of 2ⁿᵈgeneration selective and/or nonselective agonists/antagonists. Development of a microplate ligand binding assay using the PK/PBAN pheromone gland receptor was also carried out. The assay will enable screening, including high throughput, of various libraries (chemical, molecular & natural product) for the discovery of receptor specific agonists/antagonists. In summary, the body of work achieves several key milestones and brings us significantly closer to the development of novel, environmentally friendly pest insect management agents based on insect PK/PBANNPs capable of disrupting critical NP-regulated functions.
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Asvapathanagul, Pitiporn, Leanne Deocampo, and Nicholas Banuelos. Biological Hydrogen Gas Production from Food Waste as a Sustainable Fuel for Future Transportation. Mineta Transportation Institute, July 2022. http://dx.doi.org/10.31979/mti.2021.2141.
Full textAbstract:
In the global search for the right alternative energy sources for a more sustainable future, hydrogen production has stood out as a strong contender. Hydrogen gas (H2) is well-known as one of the cleanest and most sustainable energy sources, one that mainly yields only water vapor as a byproduct. Additionally, H2 generates triple the amount of energy compared to hydrocarbon fuels. H2 can be synthesized from several technologies, but currently only 1% of H2 production is generated from biomass. Biological H2 production generated from anaerobic digestion is a fraction of the 1%. This study aims to enhance biological H2 production from anaerobic digesters by increasing H2 forming microbial abundance using batch experiments. Carbon substrate availability and conversion in the anaerobic processes were achieved by chemical oxygen demand and volatile fatty acids analysis. The capability of the matrix to neutralize acids in the reactors was assessed using alkalinity assay, and ammonium toxicity was monitored by ammonium measurements. H2 content was also investigated throughout the study. The study's results demonstrate two critical outcomes, (i) food waste as substrate yielded the highest H2 gas fraction in biogas compared to other substrates fed (primary sludge, waste activated sludge and mixed sludge with or without food waste), and (ii) under normal operating condition of anaerobic digesters, increasing hydrogen forming bacterial populations, including Clostridium spp., Lactococcus spp. and Lactobacillus spp. did not prolong biological H2 recovery due to H2 being taken up by other bacteria for methane (CH4) formation. Our experiment was operated under the most optimal condition for CH4 formation as suggested by wastewater operational manuals. Therefore, CH4-forming bacteria possessed more advantages than other microbial populations, including H2-forming groups, and rapidly utilized H2 prior to methane synthesis. This study demonstrates H2 energy renewed from food waste anaerobic digestion systems delivers opportunities to maximize California’s cap-and-trade program through zero carbon fuel production and utilization.
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Asvapathanagul, Pitiporn, Leanne Deocampo, and Nicholas Banuelos. Biological Hydrogen Gas Production from Food Waste as a Sustainable Fuel for Future Transportation. Mineta Transportation Institute, July 2022. http://dx.doi.org/10.31979/mti.2022.2141.
Full textAbstract:
In the global search for the right alternative energy sources for a more sustainable future, hydrogen production has stood out as a strong contender. Hydrogen gas (H2) is well-known as one of the cleanest and most sustainable energy sources, one that mainly yields only water vapor as a byproduct. Additionally, H2 generates triple the amount of energy compared to hydrocarbon fuels. H2 can be synthesized from several technologies, but currently only 1% of H2 production is generated from biomass. Biological H2 production generated from anaerobic digestion is a fraction of the 1%. This study aims to enhance biological H2 production from anaerobic digesters by increasing H2 forming microbial abundance using batch experiments. Carbon substrate availability and conversion in the anaerobic processes were achieved by chemical oxygen demand and volatile fatty acids analysis. The capability of the matrix to neutralize acids in the reactors was assessed using alkalinity assay, and ammonium toxicity was monitored by ammonium measurements. H2 content was also investigated throughout the study. The study's results demonstrate two critical outcomes, (i) food waste as substrate yielded the highest H2 gas fraction in biogas compared to other substrates fed (primary sludge, waste activated sludge and mixed sludge with or without food waste), and (ii) under normal operating condition of anaerobic digesters, increasing hydrogen forming bacterial populations, including Clostridium spp., Lactococcus spp. and Lactobacillus spp. did not prolong biological H2 recovery due to H2 being taken up by other bacteria for methane (CH4) formation. Our experiment was operated under the most optimal condition for CH4 formation as suggested by wastewater operational manuals. Therefore, CH4-forming bacteria possessed more advantages than other microbial populations, including H2-forming groups, and rapidly utilized H2 prior to methane synthesis. This study demonstrates H2 energy renewed from food waste anaerobic digestion systems delivers opportunities to maximize California’s cap-and-trade program through zero carbon fuel production and utilization.