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Artykuły w czasopismach na temat "Enzyme assay - GPI"
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Morissette, Rachel, Yug Varma i Tamara L. Hendrickson. "Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in vivo assay". Bioscience Reports 32, nr 6 (5.10.2012): 577–86. http://dx.doi.org/10.1042/bsr20120064.
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In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.
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Novoselov, M. A., I. I. Iline, Z. Sinovcic i C. B. Phillips. "Is this imported food compliant with biosecurity regulations". New Zealand Plant Protection 67 (8.01.2014): 322. http://dx.doi.org/10.30843/nzpp.2014.67.5761.
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Imported food products can carry biosecurity hazards such as animal plant and human diseases To reduce this risk imported foods that contain ingredients of animal origin must be retorted in compliance with a New Zealand Ministry for Primary Industries (MPI) Import Health Standard AgResearch and MPI have developed a proofofconcept enzymatic colorimetric assay (Iline et al 2013; Proof of concept for a biochemical test that differentiates between heattreated and nonheattreated food products New Zealand Plant Protection 66 3439) In April 2014 MPI asked for a test to determine if a tinned food imported from India had been retorted to standard Using the proofofconcept assay all 10 samples showed weak enzyme activity while control samples heated to the MPI standard produced no enzyme activity Normally the test detects activity of the enzyme glucose phosphate isomerase (GPI) but additional testing showed that GPI was inactive A possible source of the activity was a bacterial enzyme The results suggested the product had not been retorted to the MPI standard
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Ohishi, Kazuhito, Norimitsu Inoue, Yusuke Maeda, Junji Takeda, Howard Riezman i Taroh Kinoshita. "Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins". Molecular Biology of the Cell 11, nr 5 (maj 2000): 1523–33. http://dx.doi.org/10.1091/mbc.11.5.1523.
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Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted aGAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.
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Matsuura, E., Y. Igarashi, T. Yasuda, D. A. Triplett i T. Koike. "Anticardiolipin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface." Journal of Experimental Medicine 179, nr 2 (1.02.1994): 457–62. http://dx.doi.org/10.1084/jem.179.2.457.
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Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.
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Kang, Xuedong, Alexander Szallies, Marc Rawer, Hartmut Echner i Michael Duszenko. "GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange". Journal of Cell Science 115, nr 12 (15.06.2002): 2529–39. http://dx.doi.org/10.1242/jcs.115.12.2529.
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GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.
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Dockal, Michael, Robert Pachlinger, Angelina Baldin-Stoyanova, Fabian Knofl, Nadja Ullrich, Jadranka Koehn, Hartmut J. Ehrlich i Friedrich Scheiflinger. "Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells." Blood 120, nr 21 (16.11.2012): 2210. http://dx.doi.org/10.1182/blood.v120.21.2210.2210.
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Abstract Abstract 2210 Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane. Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures: No relevant conflicts of interest to declare.
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Pingel, S., i M. Duszenko. "Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei". Biochemical Journal 283, nr 2 (15.04.1992): 479–85. http://dx.doi.org/10.1042/bj2830479.
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Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.
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Rautemaa, Riina, Gary A. Jarvis, Pertti Marnila i Seppo Meri. "Acquired Resistance of Escherichia coli to Complement Lysis by Binding of Glycophosphoinositol-Anchored Protectin (CD59)". Infection and Immunity 66, nr 5 (1.05.1998): 1928–33. http://dx.doi.org/10.1128/iai.66.5.1928-1933.1998.
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ABSTRACT Protectin (CD59) is a glycophosphoinsitol (GPI)-anchored defender of human cells against lysis by the membrane attack complex of complement. In this study, we examined whether protectin released from human cell membranes can incorporate into the surface of gram-negative bacteria. Analysis by using radiolabeled protectin, immunofluorescence, flow cytometry, and whole-cell enzyme-linked immunosorbent assay demonstrated that protectin bound to nonencapsulated Escherichia coli EH237 (Re) and EH234 (Ra) in a calcium-dependent manner. The incorporation required the GPI-phospholipid moiety since no binding of a phospholipid-free soluble form of protectin was observed. Mg2+ did not enhance the binding, and a polysialic acid capsule prevented it (strain IH3080 [O18:K1:H8]). Bound protectin inhibited the C5b-9 neoantigen expression on complement-treated bacteria. Protection against complement lysis was observed in both a colony counting assay and a bioluminescence assay, where viable EH234 bacteria expressing the luciferase gene emitted green light in the presence of the luciferine substrate. In general, two- to four-times-higher serum concentrations were needed to obtain 50% lysis of protectin-coated versus noncoated bacteria. The results indicate that protectin can incorporate in a functionally active form into the cell membranes of the two nonencapsulated deep rough E. coli strains studied.
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Chapman, J., L. Soloveichick, S. Shavit, Y. Shoenfeld i A. D. Korczyn. "Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction". Clinical and Developmental Immunology 12, nr 3 (2005): 175–80. http://dx.doi.org/10.1080/17402520500217844.
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Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (>50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.
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Peng, Li, Hong Ding, Milan Yang, Yangyan Cheng, Xiaoyan Luo, Xin Fan i Wei Jiao. "Application Value of Combined Detection of Anti-β2-GPI, ACL, and Lupus Anticoagulant in the Diagnosis of Patients with Antiphospholipid Syndrome". Evidence-Based Complementary and Alternative Medicine 2022 (3.11.2022): 1–5. http://dx.doi.org/10.1155/2022/9377334.
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Objective. To investigate the application value of combined detection of anti-beta 2-glycoprotein I antibody (anti-β2-GPI), anti-cardiolipin antibody (ACL), and lupus anticoagulant (LA) in the diagnosis of patients with antiphospholipid syndrome (APS). Methods. 30 APS patients in our hospital between Jan. 2020 and Jan. 2021 were chosen as the experimental group, and 30 healthy persons with normal physical examination during the same period were selected as the control group The anti-β2-GPI and ACL indexes of both groups were detected by enzyme-linked immunosorbent assay (ELISA), with the LA levels tested by modified dilute Russell’s viper venom time (dRVVT) and LA ratio calculated. The diagnostic efficacy of single detection and combined detection was analyzed by plotting the receiver operating characteristic (ROC) curve. Results. The serum indexes in the experimental group were remarkably higher than those in the control group ( P < 0.001 ). ROC curve analysis suggested that in the diagnosis of APS, the area under the ROC curve by detecting anti-β2-GPI, ACL, LA ratio alone and simultaneously were 0.517, 0.583, 0.683, and 0.817 respectively, and the combined detection of the three had remarkably higher sensitivity and specificity than those of each single detection. Conclusion. The indexes of anti-β2-GPI, ACL, and LA ratio were highly expressed in APS patients, and the combined detection of the three has high diagnostic value and can effectively screen and assist the diagnosis of APS.
Części książek na temat "Enzyme assay - GPI"
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Potekaev, Nikolay, Olga Zhukova i Irina Khamaganova. "False-Positive Serologic Reactions for Syphilis". W Bacterial Sexually Transmitted Infections - New Findings, Diagnosis, Treatment, and Prevention [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.106370.
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The epidemiologic situation of syphilitic infection warrants attention to diagnostic methods. Nontreponemal tests (rapid plasma regain, Venereal Disease Research Laboratory) are less reliable, as there are certain situations when false-positive reactions for syphilis antibodies may appear. Variable examinations were performed and proved that it was necessary to assess the titer of antibodies, as well as confirmation of the diagnosis by treponemal tests (fluorescent treponemal antibody, treponema pallidum hemagglutination assay, enzyme immunoassay, Western blot), were obligatory. In recent decades, new methods were elaborated (e.g., BioPlex total screen, tests with β2-GPI-dependent anticardiolipin antibody, the ARCHITECT syphilis treponema pallidum chemiluminescent immunoassay, the Elecsys immunoassay (Roche Diagnostics)). We present the review of publications on syphilis serologic diagnostics and present our own research. We did not find any mention of a false-positive test in atopic dermatitis and present a case of false-positive reactions for syphilis in such patients.
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Spickett, Gavin P. "Autoantibodies". W Oxford Handbook of Clinical Immunology and Allergy, 473–532. Oxford University Press, 2013. http://dx.doi.org/10.1093/med/9780199603244.003.0018.
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Introduction Techniques: overview Particle agglutination assays Immunoprecipitation assays Indirect immunofluorescence Direct immunofluorescence Radio-immunoassay (RIA) Enzyme-linked (EIA) and fluorescent immunoassays (FIA) Immunoblotting Acetylcholine-receptor antibodies (AChRAb) Actin antibodies Adrenal cortex autoantibodies Amphiphysin antibodies Anti-nuclear antibodies (ANA) and ANCA Aquaporin antibodies Auerbach’s plexus antibodies β2-GPI antibodies C1q antibodies...
Streszczenia konferencji na temat "Enzyme assay - GPI"
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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.