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Gotowa bibliografia na temat „Enzyme assay - PKM1/2”

Autor: Grafiati

Data publikacji: 7 września 2023

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Artykuły w czasopismach na temat "Enzyme assay - PKM1/2"

Zhan, Minglang, Xiaolei Wei, Weimin Huang, Yongqiang Wei i Ru Feng. "PKM2 Inhibition Enhances the Sensitivity of Doxorubicin in ABC Diffuse Large B-Cell Lymphoma Cells". Blood 138, Supplement 1 (5.11.2021): 4336. http://dx.doi.org/10.1182/blood-2021-146371.

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Abstract Background: Pyruvate kinase muscle isoenzyme 2 (PKM2) is a key enzyme in aerobic glycolysis and thought to contribute to cancer cell metabolic reprogramming and regulating the reactive oxygen species (ROS). Doxorubicin has been showed to induced activated-B cell types diffuse large B-cell lymphoma (ABC-DLBCL) cells death by ROS accumulation. Our purpose was to evaluate whether PKM2 inhibition could enhance the sensitivity of doxorubicin in ABC-DLBCL. Methods: MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with PKM2 inhibitor, PKM2 shRNA and doxorubicin. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green. Western Blot was used to evaluated the expression of PARP, Mcl1, Bcl2, Bax, Bim, p38 and JNK in ABC-DLBCL cells treated with PKM2 inhibition, PKM2 shRNA and doxorubicin. Results: PKM2 expression was found in both U2932 and SuDHL2 cell lines. Both PKM2 inhibitor and doxorubicin could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. PKM2 inhibitor could enhance the doxorubicin-induced apoptosis. ShRNA was used to knock down the PKM2 expression in ABC-DLBCL cell lines and PKM2 KD cell lines were more sensitive to doxorubicin. PKM2 inhibition could increase the expression of cleaved PARP, Bax, Bim, p38 and JNK as well as decrease Mcl1 and Bcl2 expression Conclusions: PKM2 inhibition could sensitize ABC-DLBCL cell lines to the cytotoxic effects of doxorubicin. Key words: PKM2, Doxorubicin, Diffuse large B cell lymphoma Disclosures No relevant conflicts of interest to declare.

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Luker, Kathryn E., i Gary D. Luker. "The CXCL12/CXCR4/ACKR3 Signaling Axis Regulates PKM2 and Glycolysis". Cells 11, nr 11 (28.05.2022): 1775. http://dx.doi.org/10.3390/cells11111775.

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In response to CXCL12, CXCR4 and ACKR3 both recruit β-arrestin 2, regulating the assembly of interacting proteins that drive signaling and contribute to the functions of both receptors in cancer and multiple other diseases. A prior proteomics study revealed that β-arrestin 2 scaffolds pyruvate kinase M2 (PKM2), an enzyme implicated in shifting cells to glycolytic metabolism and poor prognosis in cancer. We hypothesized that CXCL12 signaling regulates PKM2 protein interactions, oligomerization, and glucose metabolism. We used luciferase complementation in cell-based assays and a tumor xenograft model of breast cancer in NSG mice to quantify how CXCR4 and ACKR3 change protein interactions in the β-arrestin-ERK-PKM2 pathway. We also used mass spectrometry to analyze the effects of CXCL12 on glucose metabolism. CXCL12 signaling through CXCR4 and ACKR3 stimulated protein interactions among β-arrestin 2, PKM2, ERK2, and each receptor, leading to the dissociation of PKM2 from β-arrestin 2. The activation of both receptors reduced the oligomerization of PKM2, reflecting a shift from tetramers to dimers or monomers with low enzymatic activity. Mass spectrometry with isotopically labeled glucose showed that CXCL12 signaling increased intermediate metabolites in glycolysis and the pentose phosphate pathway, with ACKR3 mediating greater effects. These data establish how CXCL12 signaling regulates PKM2 and reprograms cellular metabolism.

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Darwish, Ibrahim, Samy Emara, Hassan Askal, Nawal El-Rabbat, Toshifumi Akizawa i Masanori Yoshiokab. "Enzyme-linked immunosorbent assay for 2-deoxycytidine". Analytica Chimica Acta 404, nr 2 (styczeń 2000): 179–86. http://dx.doi.org/10.1016/s0003-2670(99)00701-1.

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Gonzalez, Isabel, Rosario Martin, Teresa Garcia, Paloma Morales, Bernabe Sanz i Pablo Hernandez. "Antibody Sandwich Enzyme-Linked lmmunosorbent Assay". Journal of Dairy Science 77, nr 12 (grudzień 1994): 3552–57. http://dx.doi.org/10.3168/jds.s0022-0302(94)77298-2.

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Volland, Hervé, Brigitte Vulliez Le Normand, Suzanne Mamas, Jacques Grassi, Christophe Créminon, Eric Ezan i Philippe Pradelles. "Enzyme immunometric assay for leukotriene C4". Journal of Immunological Methods 175, nr 1 (wrzesień 1994): 97–105. http://dx.doi.org/10.1016/0022-1759(94)90335-2.

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Matsumoto, Akiko, Toshihiro Ino, Mitsuhiro Ohta, Tetsuya Otani, Sachiko Hanada, Atsushi Sakuraoka, Akane Matsumoto, Masayoshi Ichiba i Megumi Hara. "Enzyme-linked immunosorbent assay of nicotine metabolites". Environmental Health and Preventive Medicine 15, nr 4 (8.01.2010): 211–16. http://dx.doi.org/10.1007/s12199-009-0129-2.

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Klein, Gérard, i Jean-Louis Reymond. "An Enzyme Assay Using pM". Angewandte Chemie 113, nr 9 (4.05.2001): 1821–23. http://dx.doi.org/10.1002/1521-3757(20010504)113:9<1821::aid-ange18210>3.0.co;2-p.

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Klein, Gérard, i Jean-Louis Reymond. "An Enzyme Assay Using pM". Angewandte Chemie International Edition 40, nr 9 (4.05.2001): 1771–73. http://dx.doi.org/10.1002/1521-3773(20010504)40:9<1771::aid-anie17710>3.0.co;2-m.

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Osawa, Hisao, Olivera Josimovic-Alasevic i Tibor Diamantstein. "Enzyme-linked immunosorbent assay of mouse interleukin-2 receptors". Journal of Immunological Methods 92, nr 1 (sierpień 1986): 109–15. http://dx.doi.org/10.1016/0022-1759(86)90510-7.

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TAKAISHI, Masatoshi, Mitoshi AKIYAMA, Tomonori HAYASHI, Yuko HIRAI, Yoshie MURAKAMI, Ryuzo UEDA, Kaori TATSUGAWA, Michio YAMAKIDO i Tokuo TSUBOKURA. "ASSAY FOR THE SOLUBLE INTERLEUKIN-2 RECEPTOR BY SANDWICH ENZYME LINKED IMMUNOSORBENT ASSAY". Japanese Journal of Medical Science and Biology 43, nr 5 (1990): 151–61. http://dx.doi.org/10.7883/yoken1952.43.151.

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Rozprawy doktorskie na temat "Enzyme assay - PKM1/2"

Huang, Xinyi. "Enzymatic Characterization of N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside Deacetylase (MshB)". Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/50947.

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Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH). MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification. Therefore, MSH is considered a potential drug target. The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins). In this dissertation, we present detailed functional analysis of MshB. Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including metal ion availability and oxidative conditions. MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate. Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products. A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate. Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB. Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates. Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment.
Ph. D.

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Cruz, Taís Fukuta da [UNESP]. "Padronização e aplicação da técnica de ELISA (Enzyme-linked immunosorbent assay) indireto com anticorpo de captura para a detecção de anticoepos contra o cicovírus suíno tipo 2". Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/106028.

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Made available in DSpace on 2014-06-11T19:35:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-05Bitstream added on 2014-06-13T19:45:15Z : No. of bitstreams: 1 cruz_tf_dr_botfmvz.pdf: 667109 bytes, checksum: b54ec5fc8ecf8d9aaadfe47b8708a295 (MD5)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A quantificação de anticorpos é muito importante para monitoramento sorológico em granjas e associação com proteção contra o circovírus suíno tipo 2 (PCV2). Dessa forma este trabalho teve como objetivo padronizar e aplicar ELISA indireto com anticorpo de captura e utilizá-lo na quantificação de anticorpos contra o PCV2 em soros de suínos. Na padronização, os soros teste e os imunoreagentes básicos, incluindo, anticorpo de coelho anti-PCV2 purificado utilizado como captura e suspensão viral tiveram suas concentrações de uso ótimas determinadas. Aplicou-se o índice ELISA (IE) nos resultados dos soros de suínos para correção de variações entre testes. O teste mostrou-se específico do ponto de vista analítico e com alta reprodutibilidade podendo adequadamente ser utilizado na quantificação de anticorpos em soros de suínos contra o PCV2. Na aplicação, um total de 138 amostras de soros foi testado sendo cinco de porcas na gestação e 133 de leitões nascidos dessas porcas, acompanhados nas fases de maternidade ao crescimento em uma granja comercial com SMDS. Na avaliação da resposta de anticorpos contra o vírus, houve uma diminuição dos anticorpos da maternidade para a creche, coincidindo com o declínio dos anticorpos de origem materna e com a ocorrência da soroconversão simultaneamente com a detecção de PCV no sangue total pela PCR quantitativa. Em dois leitões com alta carga relativa de DNA viral e sinais clínicos sugestivos de SMDS, a quantidade de anticorpos detectada foi extremamente baixa
Antibody quantification is important for serological monitoring in swine herds and association with protection against porcine circovirus type 2 (PCV2). The aim of this work was to standardize and apply indirect trapping ELISA and use it in the quantification of antibodies against PCV2 in sera from pigs. The immunoreagents, including rabbit antibody anti-purified PCV2 used as trapping antibody and viral suspension had their optimum use dilution previously determined. The absorbance index (ELISA Index, EI), was used to determine the antibody concentration in pig serum directed against PCV2. The test showed high analytical specificity and reproducibility. A total of 138 serum samples was tested including five sows in gestation and 133 piglets accompanied by phases from lactation to growth in a commercial swine herd where PCV2 was previously detected. In anti PCV2 antibody response it was observed a decreasing in the phases of lactation to nursery due to decline of maternal antibodies. The seroconversion was concurrent with PCV DNA detection by quantitative PCR in total blood. In two piglets with high relative DNA viral load and compatible clinical signs of PMWS, the anti PCV2 antibody concentration was very low

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Cruz, Taís Fukuta da. "Padronização e aplicação da técnica de ELISA ("Enzyme-linked immunosorbent assay") indireto com anticorpo de captura para a detecção de anticoepos contra o cicovírus suíno tipo 2 /". Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/106028.

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Orientador: João Pessoa Araújo Junior
Banca: Alexandre Secorun Borges
Banca: Alice Fernandes Alfieri
Banca: Hélio Jose Montassier
Banca: Marcos Bryan Heinemann
Resumo: A quantificação de anticorpos é muito importante para monitoramento sorológico em granjas e associação com proteção contra o circovírus suíno tipo 2 (PCV2). Dessa forma este trabalho teve como objetivo padronizar e aplicar ELISA indireto com anticorpo de captura e utilizá-lo na quantificação de anticorpos contra o PCV2 em soros de suínos. Na padronização, os soros teste e os imunoreagentes básicos, incluindo, anticorpo de coelho anti-PCV2 purificado utilizado como captura e suspensão viral tiveram suas concentrações de uso ótimas determinadas. Aplicou-se o índice ELISA (IE) nos resultados dos soros de suínos para correção de variações entre testes. O teste mostrou-se específico do ponto de vista analítico e com alta reprodutibilidade podendo adequadamente ser utilizado na quantificação de anticorpos em soros de suínos contra o PCV2. Na aplicação, um total de 138 amostras de soros foi testado sendo cinco de porcas na gestação e 133 de leitões nascidos dessas porcas, acompanhados nas fases de maternidade ao crescimento em uma granja comercial com SMDS. Na avaliação da resposta de anticorpos contra o vírus, houve uma diminuição dos anticorpos da maternidade para a creche, coincidindo com o declínio dos anticorpos de origem materna e com a ocorrência da soroconversão simultaneamente com a detecção de PCV no sangue total pela PCR quantitativa. Em dois leitões com alta carga relativa de DNA viral e sinais clínicos sugestivos de SMDS, a quantidade de anticorpos detectada foi extremamente baixa
Abstract: Antibody quantification is important for serological monitoring in swine herds and association with protection against porcine circovirus type 2 (PCV2). The aim of this work was to standardize and apply indirect trapping ELISA and use it in the quantification of antibodies against PCV2 in sera from pigs. The immunoreagents, including rabbit antibody anti-purified PCV2 used as trapping antibody and viral suspension had their optimum use dilution previously determined. The absorbance index (ELISA Index, EI), was used to determine the antibody concentration in pig serum directed against PCV2. The test showed high analytical specificity and reproducibility. A total of 138 serum samples was tested including five sows in gestation and 133 piglets accompanied by phases from lactation to growth in a commercial swine herd where PCV2 was previously detected. In anti PCV2 antibody response it was observed a decreasing in the phases of lactation to nursery due to decline of maternal antibodies. The seroconversion was concurrent with PCV DNA detection by quantitative PCR in total blood. In two piglets with high relative DNA viral load and compatible clinical signs of PMWS, the anti PCV2 antibody concentration was very low
Doutor

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Kim, Hee Joo. "Development and application of polyclonal and monoclonal antibody based enzyme-linked immunosorbent assays for the analysis of neonicotinoid insecticides imidacloprid and thiamethoxam". Thesis, University of Hawaii at Manoa, 2003. http://proquest.umi.com/pqdweb?index=0&did=765084661&SrchMode=1&sid=5&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1233166524&clientId=23440.

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Kyllönen, H. (Heli). "Gelatinases, their tissue inhibitors and p53 in lymphomas". Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514291319.

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Abstract Lymphomas are a heterogeneous group of malignancies, which usually have a good prognosis and high cure rates. Lymphomas are sensitive to chemotherapy and radiotherapy, and many patients can be cured even after a relapse, resulting in a need for effective follow-up. However, the cost-benefit ratio of radiological imaging in predicting the forthcoming relapses is poor. Consequently, there is a need for biological prognostic and predictive markers to distinguish patients at the highest risk of relapse at the time of diagnosis or during follow-up. Despite rapid progress in lymphoma treatments, some patients still die from lymphoma. Thus, more data on the basic biological features of lymphomas are also needed. Gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) have been found to play a role in the progression of solid tumours. TP53 is a tumour suppressor gene, the mutations and protein over-expression of which have been demonstrated to be associated with survival in most cancer types. There is also some evidence that these proteins could have prognostic significance in lymphomas as well. In the present study, the tissue expression, plasma concentrations and clinical value of gelatinases and their tissue inhibitors were evaluated in lymphomas. 249 primary tissue samples from patients with Hodgkin, follicular, or diffuse large B-cell lymphoma were analysed for expression of gelatinases and/or their inhibitors using immunohistochemistry. In follicular lymphoma, p53 protein expression was also investigated. The plasma samples of 126 lymphoma patients and a control group of 44 healthy volunteers were collected and studied by ELISA. TIMP-1 expression correlated with bulky tumour and nodular sclerosis subtype of Hodgkin lymphoma. In follicular lymphoma, p53 over-expression was an independent adverse prognostic factor for survival and a predictor of histological transformation. Plasma MMP-2-TIMP-2 complex appeared to be a potential follow-up marker predicting the risk of relapse in lymphoma patients. Plasma levels of the MMP-2-TIMP-2 complex, proMMP-2, TIMP-2 and proMMP-2/TIMP-2 ratio were at abnormal levels both in patients with newly diagnosed lymphoma and those in remission compared to healthy controls. The clinical significance of these markers needs further studies.

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Honkavuori-Toivola, M. (Maria). "The prognostic role of matrix metalloproteinase-2 and -9 and their tissue inhibitor-1 and -2 in endometrial carcinoma". Doctoral thesis, Oulun yliopisto, 2014. http://urn.fi/urn:isbn:9789526204505.

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Abstract Endometrial carcinoma is the most common gynegologic malignancy in developed countries. Due to early symptoms, including abnormal uterine bleeding, endometrial cancer is often diagnosed at an early stage and in that case usually has a good prognosis and high cure rates. However, the nature of the disease is heterogeneous. During the last decades, the improvement in survival rates among endometrial cancer patients has not been significant, suggesting that the traditional clinicopathological factors may be inadequate to identify patients with high-risk disease. Furthermore, aggressive adjuvant treatments can be costly and very toxic. Therefore, better prognostic markers associated with biological aggressiveness of endometrial carcinoma are needed to identify the patients with high-risk disease, and to be able to select the treatment more individually. Gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) have been found to play a role in tumor progression. In the present work, the expression and prognostic value of MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed in endometrial carcinoma. The patient material consisted of a total of 266 women diagnosed with primary endometrial carcinoma. The tissue expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of the proteins were quantitatively measured by ELISA. Tissue MMP-2 expression associated with a worsened prognosis, whereas tissue TIMP-2 overexpression was an indicator of a favorable outcome. Furthermore, we observed a combination of strong MMP-2 and weak TIMP-2 tissue expression to identify a group of women at high risk of adverse outcome in endometrial carcinoma. Patients with negative MMP-2 immunostaining had the best prognosis, regardless of TIMP-2 staining result. In serum measurements, high preoperative TIMP-1 concentration was a prognostic indicator of unfavorable outcome. These results indicate that tissue MMP-2 and TIMP-2 as well as circulating TIMP-1 may be prognostic markers in endometrial carcinoma. Of these, tissue MMP-2 seems to be the most potent prognostic marker. Studies with larger patient materials are needed to further explore the value of these enzymes in clinical practice in endometrial cancer
Tiivistelmä Kohdunrungon syöpä on yleisin gynekologinen maligniteetti kehittyneissä maissa. Varhaisten oireiden, kuten poikkeavan verisen vuodon, vuoksi kohdunrungon syöpä havaitaan usein varhaisessa vaiheessa, jolloin sen ennuste on hyvä. Taudin käyttäytyminen voi kuitenkin olla moninaista. Viime vuosikymmenten aikana kohdunrungon syöpään sairastuneiden ennuste ei ole merkittävästi parantunut. Vaikuttaisi siltä, että perinteiset ennustetekijät eivät ole riittävän tarkkoja ennustamaan syövän taudinkulkua. Lisäksi liitännäishoidot voivat olla kalliita, ja niihin voi liittyä vakavia haittavaikutuksia. Uusien biologisten ennustetekijöiden löytäminen olisi tärkeää, jotta aggressiivista syöpätyyppiä sairastavat potilaat pystyttäisiin tunnistamaan entistä paremmin, ja hoito kyettäisiin räätälöimään yksilöllisemmin taudinkuvaa vastaavasti. Gelatinaasien (MMP-2 ja MMP-9) sekä niiden kudosinhibiittoreiden (TIMP-1 ja TIMP-2) on havaittu osallistuvan syövän etenemiseen. Tässä tutkimuksessa tarkasteltiin MMP-2:n ja MMP-9:n sekä niiden kudosinhibiittoreiden TIMP-1:n ja TIMP-2:n ilmentymistä ja ennusteellista merkitystä kohdunrungon syövässä. Aineisto käsitti yhteensä 266 primaariseen kohdunrungon syöpään sairastunutta naista. Määritysmenetelminä käytettiin sekä immunohistokemiallista värjäystä parafiiniin valettujen kudosnäytteiden osalta että ELISA-määrityksiä ennen hoitoa otettujen seeruminäytteiden osalta. Syöpäkudoksen runsas MMP-2 -proteiinin ilmentyminen liittyi epäsuotuisaan ennusteeseen, kun taas kasvainkudoksen voimakas TIMP-2 -proteiinin ilmentyminen oli hyvän ennusteen merkki. Lisäksi kasvainkudoksen voimakkaan MMP-2- ja heikon TIMP-2 -proteiinien ilmentymisen yhdistelmän havaittiin liittyvän suurempaan syövästä johtuvaan kuolleisuuteen. MMP-2 -negatiivisten potilaiden eloonjäämisennuste oli paras, TIMP-2 -värjäystuloksesta riippumatta. Seerumin korkea TIMP-1 -pitoisuus oli merkittävä huonontuneen ennusteen merkki. Tutkimuksen tulokset viittaavat siihen, että kasvainkudoksessa esiintyvät MMP-2- ja TIMP-2 -proteiinit samoin kuin seerumin TIMP-1 -pitoisuus voivat ennustaa kohdunrungon syövän kliinistä käyttäytymistä. Kasvainkudoksessa esiintyvä MMP-2 -proteiini vaikuttaisi olevan merkittävin ennusteellinen tekijä, mutta tulosten varmistamiseksi tarvitaan lisää tutkimuksia suuremmilla potilasaineistoilla

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Muchindu, Munkombwe. "Electrochemical ochratoxin a immunosensors based on polyaniline nanocomposites templated with amine- and sulphate-functionalised polystyrene latex beads". Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3815_1306752491.

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Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3 &minus
) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate (PSNP-OSO3 &minus
) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 &minus
) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10&minus
8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were <
100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 &mu
A/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 &mu
M NO2 &minus
, were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 &minus
, respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors.

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chang, Kun-Chao, i 張昆照. "Development of an Enzyme-Linked Immunosorbent Assay (ELISA) For Serological Diagnosis of Porcine Circovirus Type 2". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/53674921916332084364.

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碩士
國立臺灣大學
獸醫學研究所
95
Currently, porcine circovirus 2 (PCV2) is an important emerging pathogen associated with post-weaning multisystemic wasting syndrome ( PMWS) in pig farm. This syndrome of disease occurs commonly in 5-12-week-old pigs. Clinically, PMWS is characterized by progressive weight loss, respiratory signs, and occasional the jaundice in pigs. The objective of this study was to develop a recombinant protein of the viral capsid protein (ORF2) as antigen of enzyme-linked immunosorbent assay (ELISA) kit for the detection of antibody to porcine circovirus type 2. Specific primers were synthesized according to PCV2 ORF2 DNA sequence starting from the 123 bp at 5’ end. The PCR amplicom were cloned into pMAL-c2X vector and expressed in E. Coli. The purified protein (MBP-D41) was used as ELISA antigen for detecting PCV2 antibody. To investigate the dynamics of PCV2 antibody production in relation to virus protection or circulation within the farm, we collected 50 blood samples from each farm at the following stages: 1 week, 3-4 weeks, 8 weeks, finishing pig (70-80 kg), and sow. Serological tests of PCV2 antibody were performed with commercial and “recombinant antigen (MBP-D41)” diagnostic kits in wild field. The average percentage of pigs with PCV2 antibody response in ELISA test was 90% (1 week), 47.4% (3-4 weeks), 20% (8 weeks), 97.5% (finishing pig) and 85% (sow), respectively. The distribution of the PCV2 antibody decreased from one to eight –week-old pigs. The level of antibody was almost undetectable at eight-week-old pigs. Consequently, the sensitivity to viral infection was found to be increased. There was no significant difference between our developing diagnostic kit and the commercial diagnostic kit for the detection of PCV2 antibody in various growth stages of pig. These results demonstrate that the MBP-D41 protein is a potential candidate as an ELISA antigen.

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Tang, Yi-Syuan, i 唐翊軒. "Development of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Porcine Circovirus Type 2 (PCV2) Antigen Detection". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/36287866158602127090.

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碩士
國立宜蘭大學
生物技術與動物科學系
104
Porcine circovirus-associated disease (PCVAD) is an important disease that affects the pig industry, and the major pathogen is Porcine Circovirus type 2 (PCV2). In order to cope with the heavy loss caused by PCV2, leading pharmaceutical and biotech companies have launched PCV2 vaccines and diagnostic reagents. Currently on the market, the diagnostic products for PCV2 detection aim to detect anti-PCV2 antibodies in sera. No commercial products that detect the antigen of PCV2 virus particle are available. The object of this dissertation was to develop an enzyme-linked immunosorbent assay (ELISA) to detect antigens of PCV2. Firstly, monoclonal antibodies against PCV2 virus particle were produced. A large amount of virus obtained from cell culture was purified and used to immunize mice as well as produce hybridomas. After several screenings, a hybridoma cell line, 3A10-D1-H2, capable of secreting antibodies against PCV2 virus particle was successfully isolated. The monoclonal antibodies were verified to recognize PCV2 virus particle by ELISA, indirect immunofluorescence assay (IFA), etc. Purified monoclonal antibodies obtained from murine ascites and rabbit polyclonal antibodies were used to develop an ELISA. Results showed that the sandwich ELISA successfully detected PCV2 viruses produced via cell culture and the sensitivity for purified virus reached ng/ml levels. On the other hand, the monoclonal antibodies produced from this study have the ability to neutralize PCV2 virus infection. The sequence of the high viability zone of PCV2 antibody was also revealed by nucleotide sequence analysis with the hybridoma cell line. The information paves the way for subsequent development of therapeutic chimeric antibodies.

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Manenzhe, Shumani Charlotte. "The diagnostic accuracy of the HIV 1/2/subtype O Tri-line HIV rapid test in comparison to ELISA". Thesis, 2018. https://hdl.handle.net/10539/25389.

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A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Dentistry. Johannesburg, 2018.
Background: Accurate HIV diagnosis is critical and can be life-saving. A Rapid Test (RT) is considered key to HIV prevention and management. Some studies have found RT to be comparable with ELISA whilst others have reported on lower sensitivity. Aim and study design: The aim of this retrospective comparative descriptive study was to evaluate the sensitivity and specificity of the Tri-line HIV rapid test device in comparison to ELISA on patient records from Wits Oral Health Centre (WOHC) between 2014 and 2016 Method: The study population comprised records of patients older than18 months who had Tri-Line HIV RT and blood drawn for ELISA on the same day. Descriptive analysis of the data was carried out. Results: The sensitivity of Tri-line was 80% (CI: 59-93%) and specificity was 100% (CI: 83-100%). The PPV was 100% (CI: 83-100%) and NPV was 80% (CI: 65-90%). ROC area of 0.9 at 95% CI was determined. Conclusion: Due to a low sample size in this study a definitive conclusion could not be drawn. However on the basis of the results obtained, although the tri-line RT showed lower sensitivity it was shown to be a clinically useful test.
LG2018

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Książki na temat "Enzyme assay - PKM1/2"

Hosseini, Samira, Patricia Vázquez-Villegas, Marco Rito-Palomares i Sergio O. Martinez-Chapa. Enzyme-linked Immunosorbent Assay (ELISA). Singapore: Springer Singapore, 2018. http://dx.doi.org/10.1007/978-981-10-6766-2.

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Wilson, John W., i Lynn L. Estes. Antiretroviral Therapy for HIV Infection. Oxford University Press, 2012. http://dx.doi.org/10.1093/med/9780199797783.003.0134.

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• Obtain confirmatory human immunodeficiency virus (HIV) testing by rapid test or enzyme-linked immunosorbent assay (ELISA); optimally repeat HIV viral load (VL) and CD4 T-cell (CD4) count 2 times before initiation of therapy; a substantial change in CD4 count is generally >30%• Perform VL immediately before treatment initiation (or change in therapy) and again 2–8 weeks later; for the latter, the optimal decrease would be at least 1 log...

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Gibson, K. Michael, Cornelis Jakobs i Philip L. Pearl. Succinic Semialdehyde Dehydrogenase Deficiency. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780199972135.003.0029.

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Succinic semialdehyde dehydrogenase (SSADH) deficiency presents with intellectual disability, disproportionate deficit in expressive language, hypotonia, ataxia, and seizures.1,2 (1 Pearl et al 2011; 2 Vogel et al 2012). A diagnosis of autism spectrum disorder frequently occurs, correlated with neuropsychiatric morbidity (ADHD, OCD, PDD). 1,3 The biochemical hallmark, γ‎-hydroxybutyric acid (GHB), is elevated in physiological fluids, as is γ‎-aminobutyrate (GABA) in cerebrospinal fluid (CSF).4,5 Both species are neuroactive. Clinical manifestations are universally present in early childhood, although diagnosis delayed to adulthood has been reported.6 Acute decompensation or complications relate primarily to seizures, intercurrent illnesses sometimes associated with respiratory dysfunction in the setting of hypotonia, or adverse medication responses. Diagnostic confirmation requires urine organic acid analysis (increased GHB) with confirmation via enzyme assay (white cells) and/or molecular characterization of the aldehyde dehydrogenase 5a1 (ALDH5A1) gene.

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Części książek na temat "Enzyme assay - PKM1/2"

Goldring, O. L. "DETERGENT SOLUBILISED ANTIGENS IN ENZYME IMMUNOASSAY WITH PARTICULAR REFERENCE TO ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) SYSTEMS". W Immunoassay Technology Vol. 2, redaktor S. B. Pal, 189–214. Berlin, Boston: De Gruyter, 1986. http://dx.doi.org/10.1515/9783110884692-010.

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Smeenk, R. "DETECTION OF ANTIBODIES TO DNA BY ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)". W Immunoassay Technology Vol. 2, redaktor S. B. Pal, 121–44. Berlin, Boston: De Gruyter, 1986. http://dx.doi.org/10.1515/9783110884692-007.

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Moore, A. L., i W. B. Ershler. "SPECIFIC ANTIBODY SYNTHESIS IN VITRO : AN APPRAISAL OF THE MICROCULTURE ANTIBODY SYNTHESIS ENZYME-LINKED ASSAY (MASELA)". W Immunoassay Technology Vol. 2, redaktor S. B. Pal, 105–20. Berlin, Boston: De Gruyter, 1986. http://dx.doi.org/10.1515/9783110884692-006.

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Trucksess, Mary W., Deborah K. Morris i Ernie Lewis. "Enzyme-Linked Immunosorbent Assay of Aflatoxins B1, B2, and G1 in Corn, Cottonseed, Peanuts, Peanut Butter, and Poultry Feed". W Biodeterioration Research 2, 301–11. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4684-5670-7_27.

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Itak, Jeanne A., William A. Day, Angel Montoya, Juan J. Manclús, Amy M. Phillips, Dwayne A. Lindsay i David P. Herzog. "A Paramagnetic Particle-Based Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of 3,5,6-Trichloro-2-pyridinol in Water". W ACS Symposium Series, 261–70. Washington, DC: American Chemical Society, 1997. http://dx.doi.org/10.1021/bk-1997-0657.ch021.

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Tahseen, Rabia, Mohammad Parvez, G. Sravan Kumar i Parveen Jahan. "Prognostic Importance of Th1:Th2 (IL-1β/IL-10) Cytokine Ratio in Adult Onset-Bronchial Asthma". W Proceedings of the Conference BioSangam 2022: Emerging Trends in Biotechnology (BIOSANGAM 2022), 176–87. Dordrecht: Atlantis Press International BV, 2022. http://dx.doi.org/10.2991/978-94-6463-020-6_18.

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AbstractBronchial asthma is a complex respiratory disorder, exhibits several endotypes and phenotypes due to different underlying cellular and molecular mechanisms. Globally it affects 300 million individuals, with the prevalence of 2–3% in India, contributing to morbidity and mortality. Over 50 cytokines have been identified in asthma. The dysregulation in Th1 and Th2 cytokines is implicated in the patho-mechanism of pulmonary inflammation and airway remodeling. The aim of the current study was to access the circulating levels of IL-1β (pro-inflammatory) and IL-10 (anti-inflammatory) cytokines using sandwich enzyme-linked immunosorbent assay (ELISA). In this case-control study we recruited a total of 164 subjects (104 adult onset asthma patients and 60 non-asthmatic healthy controls) from south India. Data exhibited increased levels of IL-1β and decreased levels of IL-10 in asthma patients compared to the healthy controls. Subgroup analysis revealed significant elevation in the circulating levels of IL-1β and Th1:Th2 (IL-1β/IL-10) ratio in patients with uncontrolled and long-standing disease (>10 years). Receiver operating curve analysis of individual cytokines and ratios showed good and excellent discriminating capacity respectively for health vs disease and controlled vs uncontrolled. However, IL-1β showed better incisive capacity for disease duration. Based on our observation it appears that rather than individual cytokine(s), the balance between pro and anti-inflammatory cytokines are crucial in the patho-mechanism of asthma. However, developing a signature profile of multiple cytokines using cut-off values may prove to be more promising for diagnostic, prognostic and therapeutic purposes of bronchial asthma.

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Giri, Basant. "Microfluidic Enzyme-Linked Immunosorbent Assay". W Laboratory Methods in Microfluidics, 121–28. Elsevier, 2017. http://dx.doi.org/10.1016/b978-0-12-813235-7.00019-2.

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Tantray, Javeed Ahmad, Sheikh Mansoor, Rasy Fayaz Choh Wani i Nighat Un Nissa. "Enzyme activity using starch assay". W Basic Life Science Methods, 79–81. Elsevier, 2023. http://dx.doi.org/10.1016/b978-0-443-19174-9.00019-2.

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Drijvers, Jefte M., Imad M. Awan, Cory A. Perugino, Ian M. Rosenberg i Shiv Pillai. "The Enzyme-Linked Immunosorbent Assay". W Basic Science Methods for Clinical Researchers, 119–33. Elsevier, 2017. http://dx.doi.org/10.1016/b978-0-12-803077-6.00007-2.

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Lee, L. James, Shang‐Tiang Yang, Siyi Lai, Yunling Bai, Wei‐Cho Huang i Yi‐Je Juang. "Microfluidic Enzyme‐Linked Immunosorbent Assay Technology". W Advances in Clinical Chemistry, 255–95. Elsevier, 2006. http://dx.doi.org/10.1016/s0065-2423(06)42007-2.

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Streszczenia konferencji na temat "Enzyme assay - PKM1/2"

Moore, J., L. Robertson, T. Ferguson, E. Cain, J. Goodman, C. Colley, M. Orr i T. Moore. "Clinical performance evaluation of an anti-SARS-CoV-2 IgG enzyme-linked immunosorbent assay". W ERS International Congress 2022 abstracts. European Respiratory Society, 2022. http://dx.doi.org/10.1183/13993003.congress-2022.4612.

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Sugihara, T., J. Takamatsu, T. Kamiya, H. Saito, K. Kimata i K. Kato. "A SENSITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY(ELISA) FOR SERUM LAMININ". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643554.

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Laminin, a large glycoprotein, is a major and specific component of basement membrane. There were little or no circulating laminin in normal persons, although some recent reports have showed increased values in various diseases(ex. diabetes mellitus and liver disease) by a radioimmunoassay(RIA) using laminin fragment. The minimum detectable sensitivity of RIA was reported to be 20 ng/ml of serum sample. We describe here a more sensitive immunoassay system, and also the concentrations of laminin in sera from healthy subjects and patients. A sandwich ELISA method for measurement of laminin was established by use of purified antibodies to mouse laminin. The assay system consisted of poly-stylene balls with immobilized antibody F(ab’)2 fragments and the same antibody Fab1 fragments labeled with β-D-galactosidase from E.Coli. The assay was highly sensitive and can detect as small as 0.5 ng/ml of serum laminin.Coefficients of variation in within-run and between-run precision studies for serum laminin were good. Serum laminin levels in healthy subjects of various ages ranged from 1.5 to 3.9 ng/ml(n=60). Fifty eight patient sera (collagen disease(n=18), hepatic disease(n=20), and renal disease (n=20)) were examined. Significant differences between the normal sera and 3 diseases sera were observed as shown belowIt is concluded that circulating laminin apparently exists in normal persons and there are higher laminin level in some diseases which appears to involve basement membrane-rich organ

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HUSSAIN, Zainab Khidhair, i Bushra Rashid Ibrahim. "COMPARISON BETWEEN CARDIAC ENZYMES IN PATIENTS WITH HYPOTHYROIDISM AND HYPERTHYROIDISM". W III.International Scientific Congress of Pure,Appliedand Technological Sciences. Rimar Academy, 2021. http://dx.doi.org/10.47832/minarcongress3-2.

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Thyroid hormones modify each cardiovascular system component, it's essential for the function and development off the cardiac system. Thyroid hormones and the cardiac enzyme were measured in (120) Iraqi women aged (20-65) years in three groups: patients with hypothyroidism, hyperthyroidism, and control. Thyroid hormones (TSH, T3, and T4) were measure by ELISA by using a procedure of TOSOH,CHINA, also, cardiac enzymes were determined by biochemical assay of Biosystem company, Barcelona. The results showed the level of CK enzyme increasing non significantly (53.61) between groups in hyperthyroidism (G1), hypothyroidism(G2) and was (151.40 ±8.86uk) and (127.80 ±21.82uk) respectively compared with control was (G3) (100.60 ±18.80 uk),also the level of Troponin- I enzyme increasing non significantly (213.42) between groups was in(G1) (430.20 ±53.38) (Pg/UL), (G2) was (369.20 ±75.75) (Pg/UL) and (G3) (275.60 ±76.18).In comparison the study showed decreasing non significantly in cardiac enzymes as AST and ALT. It concluded that non-significant effect of thyroid hormones on the level of cardiac enzyme in both patients with hypothyroidism and hyperthyroidism. Key words: Hyperthyroidism, Cardiac Enzyme, Hypothyroidism, Hormones, Troponin I.

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Kostina, J., i E. Tarasova. "INTERPRETATION OF THE RESULTS OF AN ENZYME – LINKED IMMUNOSORBENT ASSAY IN THE DIAGNOSIS OF HERPES VIRUS". W SAKHAROV READINGS 2020: ENVIRONMENTAL PROBLEMS OF THE XXI CENTURY. Minsk, ICC of Minfin, 2020. http://dx.doi.org/10.46646/sakh-2020-2-84-86.

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Moore, J., L. Robertson, T. Ferguson, E. Cain, J. Goodman, C. Colley, M. Orr i T. Moore. "P166 An evaluation of the clinical characteristics of an Anti-SARS-CoV-2 IgG enzyme-linked immunosorbent assay". W British Thoracic Society Winter Meeting 2022, QEII Centre, Broad Sanctuary, Westminster, London SW1P 3EE, 23 to 25 November 2022, Programme and Abstracts. BMJ Publishing Group Ltd and British Thoracic Society, 2022. http://dx.doi.org/10.1136/thorax-2022-btsabstracts.300.

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MacGregor, I. R., N. A. Booth, N. R. Hunter i B. Bennet. "QUANTIFICATION OF ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) ANTIGEN BY AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644450.

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An ELISA to measure PAI-1 antigen has been developed using PAI-1 purified from human endothelial cell conditioned medium and a monospecific antiserum raised against it in the rabbit. Test and standard samples diluted in assay buffer containing non-immune rabbit serum were incubated in microplate wells coated with anti-PAI-1 IgG. Then biotinylated anti-PAI-1 IgG was added to the wells followed by a streptavidin-biotinylated horseradish peroxidase complex. Tetramethylbenzidine was used as substrate and the optimised ELISA had a detection limit of 0.2 ng PAI-1 ml-1 sample, using purified PAI-1 of known concentration as a standard for the assay.PAI-1 antigen was readily detectable in human plasmas and higher concentrations were invariably detected in the corresponding whole blood sera.α2antiplasmin and antithrombin III which have been shown to have high degrees of sequence homology with PAI-J were undetectable in the ELISA at concentrations of 10 ug ml-1 . Sera from baboon and Rhesus and Cynomologus monkeys exhibited partial cross reactivity in the ELISA while no crossreactivities were observed with a wide range of non-primate sera that included dog, goat, cow, horse, pig and rat.A variety of human cell cultures were assayed for PAI-1 antigen. Endothelial cells from umbilical cord veins and arteries and from adult sapgenous veins secreted, typically,1-2 ug PAI-1 24-1 hr per 10 cells. This represented approximately 2-4% of the total secreted protein. Detectable but considerably lower levels of PAI-1 were secreted by keratinocytes and fibroblasts as well as a lung carcinoma cell line A549.No PAI-1 was detected in media conditioned by two melanoma cell lines, a breast carcinoma cell line MCF7 or a monocytic leukaemia JIII. The ELISA proved suitable for monitoring altered cellular PAI-1 metabolism, and in conjunction with functional assays, for investigating changes in PAI-1 specific activity.

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Dutta, Debashis, i Naoki Yanagisawa. "Microfluidic Devices for Enhancing the Sensitivity of ELISA Methods". W ASME 2011 9th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2011. http://dx.doi.org/10.1115/icnmm2011-58284.

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Enzyme-linked immunosorbent assays (ELISA) are critically important tools in biological research, allowing the presence and concentrations of a wide variety of key biochemical intermediates to be determined. While the signal amplification that is the core advantage of ELISA methods is impressive, it is nevertheless the case that it is insufficient for some particularly demanding challenges in terms of sensitivity, assay time, or sample size. In this paper, we discuss three different approaches developed in our laboratory that can improve the sensitivity of ELISA methods by 2–3 orders of magnitude. Two of these approaches have been shown to reduce the minimum detectable concentration of the target analyte in the system through trapping of the analyte species and the enzyme reaction product around a semi-permeable membrane. The third approach, on the other hand, focuses on reducing the sample volume requirement in these assays by implementing multiplex ELISA methods in a single microfluidic channel using the same enzyme label. This multiplex technique relies on the slow diffusion of the enzyme reaction product across adjacent assay segments for accurate quantitation and has been demonstrated to have a limit of detection substantially better than that of commercial microtiter plates. We believe the combination of these approaches could significantly extend the applicability of the ELISA technique to more challenging assays than is currently possible.

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Liao, Uland, Armando Tovar, Philip Felgner i Abraham P. Lee. "A Microfluidic Approach and Enhancement Towards a Colorimetric Enzyme-Linked-Immunosorbant-Assay for Diagnostic Detection of Infectious Diseases". W ASME 2007 2nd Frontiers in Biomedical Devices Conference. ASMEDC, 2007. http://dx.doi.org/10.1115/biomed2007-38105.

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Accounting for more than 13 million deaths a year, infectious diseases have become the world’s biggest killer of children and young adults worldwide [1]. Diagnostic tools and technologies are vital towards identifying the presence and treatment of these diseases. Detection methods have commonly relied on DNA using polymerase-chain-reaction (PCR), however antibody methods have become popular due to growing trends in technology and detection sensitivity. ImmPORT Therapeutics, a leading group in generating infectious disease proteome microarrays, has developed multiplex systems for comprehensive analysis of immune responses to multiple infectious diseases [2]. Current microarray handling however requires conventional lab-bench methods that require whole-day processes and large amounts of user-handling confined to laboratory settings. Miniaturization of laboratory processes would provide numerous advantages in terms of cost, time, portability and multistage automation in addition to what is already offered. The proposed microfluidic device is a colorimetric enzyme-linked-immunosorbant assay (ELISA) for antibody detection of infectious agents that draws on ImmPORT Therapeutics technology with a purpose of decreasing reagent volumes and times potentially unattainable through conventional methods.

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Purdon, A. D., i J. B. Smith. "ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644628.

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Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

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Zanarel, Palomali, Marina Grigoli, Danielle de Oliveira, Patrícia Manzine i Márcia Cominetti. "IFG-1 PLASMA LEVELS ARE ALTERED IN PATIENTS WITH ALZHEIMER’S DIASEASE AND DIABTES MELLITUS TYPE 2". W XIII Meeting of Researchers on Alzheimer's Disease and Related Disorders. Zeppelini Editorial e Comunicação, 2021. http://dx.doi.org/10.5327/1980-5764.rpda045.

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Background: Insulin plays an important role in mechanisms related to brain activity and in memory formation. Alterations in the insulin pathway may be related to Alzheimer’s disease (AD) and patients with type 2 diabetes mellitus (DM2) are more likely to develop AD, compared to metabolically and cognitively healthy individuals. Objectives: To evaluate the IGF-1 levels in plasma samples from individuals with AD or DM2 isolated or with both diseases concomitantly and to compare with the levels from cognitively and metabolically healthy older adults. Methods: This is a cross-sectional, descriptive and comparative study ethically approved (CAAE: 31634720.9.0000.5504) that has been developed in the city of São Carlos with a sample of 36 participants, aged over 60 years, users of health services in the municipality. Specific inclusion and exclusion criteria and cognitive assessment instruments were applied to participants from all groups. The quantification of plasma levels of IGF-1 was performed using the ELISA (Enzyme-Linked Immunosorbent Assay) technique. Results: Participants with AD and DM concomitantly had higher levels of IGF-1 when compared with the individuals in the control group (p=0.0003) and with participants with DM (p=0.0167). Conclusions: The significant increase in IGF-1 plasma levels in the AD+DM and DM groups may indicate an initial compensatory response against neuronal damage in these patients.

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Raporty organizacyjne na temat "Enzyme assay - PKM1/2"

Hodges, Thomas K., i David Gidoni. Regulated Expression of Yeast FLP Recombinase in Plant Cells. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7574341.bard.

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Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.

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Delwiche, Michael, Boaz Zion, Robert BonDurant, Judith Rishpon, Ephraim Maltz i Miriam Rosenberg. Biosensors for On-Line Measurement of Reproductive Hormones and Milk Proteins to Improve Dairy Herd Management. United States Department of Agriculture, luty 2001. http://dx.doi.org/10.32747/2001.7573998.bard.

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The original objectives of this research project were to: (1) develop immunoassays, photometric sensors, and electrochemical sensors for real-time measurement of progesterone and estradiol in milk, (2) develop biosensors for measurement of caseins in milk, and (3) integrate and adapt these sensor technologies to create an automated electronic sensing system for operation in dairy parlors during milking. The overall direction of research was not changed, although the work was expanded to include other milk components such as urea and lactose. A second generation biosensor for on-line measurement of bovine progesterone was designed and tested. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing, and a real-time assay was developed. The biosensor was designed using micropumps and valves under computer control, and assayed fluid volumes on the order of 1 ml. An automated sampler was designed to draw a test volume of milk from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min. Progesterone could be measured at concentrations low enough to distinguish luteal-phase from follicular-phase cows. The potential of the sensor to detect actual ovulatory events was compared with standard methods of estrus detection, including human observation and an activity monitor. The biosensor correctly identified all ovulatory events during its testperiod, but the variability at low progesterone concentrations triggered some false positives. Direct on-line measurement and intelligent interpretation of reproductive hormone profiles offers the potential for substantial improvement in reproductive management. A simple potentiometric method for measurement of milk protein was developed and tested. The method was based on the fact that proteins bind iodine. When proteins are added to a solution of the redox couple iodine/iodide (I-I2), the concentration of free iodine is changed and, as a consequence, the potential between two electrodes immersed in the solution is changed. The method worked well with analytical casein solutions and accurately measured concentrations of analytical caseins added to fresh milk. When tested with actual milk samples, the correlation between the sensor readings and the reference lab results (of both total proteins and casein content) was inferior to that of analytical casein. A number of different technologies were explored for the analysis of milk urea, and a manometric technique was selected for the final design. In the new sensor, urea in the sample was hydrolyzed to ammonium and carbonate by the enzyme urease, and subsequent shaking of the sample with citric acid in a sealed cell allowed urea to be estimated as a change in partial pressure of carbon dioxide. The pressure change in the cell was measured with a miniature piezoresistive pressure sensor, and effects of background dissolved gases and vapor pressures were corrected for by repeating the measurement of pressure developed in the sample without the addition of urease. Results were accurate in the physiological range of milk, the assay was faster than the typical milking period, and no toxic reagents were required. A sampling device was designed and built to passively draw milk from the long milk tube in the parlor. An electrochemical sensor for lactose was developed starting with a three-cascaded-enzyme sensor, evolving into two enzymes and CO2[Fe (CN)6] as a mediator, and then into a microflow injection system using poly-osmium modified screen-printed electrodes. The sensor was designed to serve multiple milking positions, using a manifold valve, a sampling valve, and two pumps. Disposable screen-printed electrodes with enzymatic membranes were used. The sensor was optimized for electrode coating components, flow rate, pH, and sample size, and the results correlated well (r2= 0.967) with known lactose concentrations.

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Christopher, David A., i Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, maj 2004. http://dx.doi.org/10.32747/2004.7586534.bard.

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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.

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