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1
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.
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Liu, Qin, Xueyan Zhan, Dongfang Li, Junlong Zhao, Haiyong Wei, Heba Alzan i Lan He. "Establishment and Application of an Indirect Enzyme-Linked Immunosorbent Assay for Measuring GPI-Anchored Protein 52 (P52) Antibodies in Babesia gibsoni-Infected Dogs". Animals 12, nr 9 (6.05.2022): 1197. http://dx.doi.org/10.3390/ani12091197.
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Babesia gibsoni is a malaria-like protozoan that parasitizes the red blood cells of canids to cause babesiosis. Due to its high expression and essential function in the survival of parasites, the Glycosylphosphatidylinositol (GPI) anchor protein family is considered an excellent immunodiagnostic marker. Herein, we identified a novel GPI-anchored protein named as BgGPI52-WH with a size of 52 kDa; the recombinant BgGPI52-WH with high antigenicity and immunogenicity was used as a diagnostic antigen to establish a new iELISA method. The iELISA had a sensitivity of 1:400, and no cross-reaction with other apicomplexan parasites occurred. We further demonstrated that the degree of variation was less than 10% using the same samples from the same or different batches of an enzyme-labeled strip. It was found that the method was able to detect early infection (6 days after infection) in the sera of the B. gibsoni-infected experimental dogs in which antibody response to rBgGPI52-WH was evaluated. Clinical sera from pet hospitals were further tested, and the average positive rate was about 11.41% (17/149). The results indicate that BgGPI52-WH is a reliable diagnostic antigen, and the new iELISA could be used as a practical method for the early diagnosis of B. gibsoni.
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Kedar, Prabhakar S., Vinod Gupta, Rashmi Dongerdiye, Ashish Chiddarwar, Prashant Warang i Manisha R. Madkaikar. "Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients". Journal of Clinical Pathology 72, nr 1 (18.10.2018): 81–85. http://dx.doi.org/10.1136/jclinpath-2018-205420.
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Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic mutation is the gold standard method for confirmation of disease, but causative genes involved in CHA are numerous, and identifying a gene-by-gene approach using Sanger sequencing is also cumbersome, expensive and labour intensive. Recently, next-generation targeted sequencing is more useful in the diagnosis of unexplained haemolytic anaemia. We used targeted next-generation sequencing (NGS) clinical panel for diagnosis of unexplained haemolytic anaemia in two Indian patients which were pending for a long time. All possible causes of haemolytic anaemia were found within normal limit. NGS by clinical exome panel revealed homozygous novel missense mutation in exon 12, c.1009G>A (p.Ala337Thr) in both patients. We further confirm by measuring red blood cell GPI activity in the patients and showed deficiency whereas parents were having intermediate activity. c.1009G>A mutation was also confirmed by Sanger sequencing of exon 12 of GPI gene. The structural–functional analysis by bioinformatics software like Swiss PDB, PolyPhen-2 and PyMol suggested that this pathogenic variant has a direct impact on the structural rearrangement at the region near the active site of the enzyme. This rapid and high-performance targeted NGS assay can be configured to detect specific CHA mutations unique to an individual defect, making it a potentially valuable method for diagnosis of unexplained haemolytic anaemia.
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Peffault de Latour, Regis, Valeria Visconte, Keyvan Keyvanfar, Olga Nunez, Marie Desierto, Jichun Chen, Adrian Wiestner i Neal S. Young. "Endoplasmic Reticulum Stress Is a Target for Therapy in Paroxysmal Nocturnal Hemoglobinuria." Blood 114, nr 22 (20.11.2009): 160. http://dx.doi.org/10.1182/blood.v114.22.160.160.
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Abstract Abstract 160 Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolytic anemia, bone marrow failure and venous thromboembolism. The disease is caused by a somatic mutation of the X-linked gene PIG-A, encoding a key enzyme responsible for the biosynthesis of the glycosylphosphatidylinositol anchored proteins (GPI-APs). Unfolded GPI-APs are translated and degraded intracellularly in the endoplasmic reticulum (ER) via the proteasome machinery. We hypothesized that the accumulation of misfolded proteins in mutant PNH cells activates the unfolded protein response (UPR) signaling cascade, which physiologically maintains the quality of newly synthesized proteins. UPR could thus represent a new target for therapy in PNH. We first assessed the sensitivity of GPI-AP- deficient K562 cells to the proteasome inhibitor PS-341 (Velcade‘) by flow cytometry after staining for annexin V and propidium iodide (PI). After 24 hour exposure to PS-341, cytotoxicity was significantly higher in the K562 GPI-AP-deficient cell line compared to their wild type counterpart at all dose tested (0 to 200nM). At 50nM, the early stage apoptosis was 21±2% in the K562 GPI-AP-deficient cell line versus 9.3±2% for their wild type counterpart (p=0.013) while the late stage apoptosis was 38±2.3% versus 22±2.3% (p<10−3), respectively. Similar results were obtained in immortalized lymphoblastoid cells derived from a PNH patient after PS-341 exposure. We reproduced those results with 2 other proteasome inhibitors (MG132 and Lactacystine) confirming the fundamental role of the proteasome in PNH. By immunoblot, K562 GPI-AP-deficient cell line exhibited more expression of chaperone proteins (CHOP and BiP/GRP78) compared to wild type cells after PS-341 exposure; evidence of the UPR is initiated by accumulation of unfolded proteins. UPR activation was confirmed by the detection of XBP1 splicing form in K562 GPI-AP-deficient cells by polymerase chain reaction (PCR). Moreover, PS-341 promoted pro-apoptotic/terminal UPR gene expression in K562 GPI-APs deficient cells as illustrated by the increase expression of an UPR specific pro-apoptotic protein, NOXA. We observed a concomitant arrest in the G2 phase of the cell cycle, as detected by flow cytometry after staining with PI. We employed a mouse model bearing a conditional Pig-a gene deletion in hematopoietic cells. When the animals were treated with PS-341 (15 ug/day once a week, intraperitoneally), there was a significantly higher proportion of apoptotic GPI-AP-deficient B cells compared to normal B cells in early stage apoptosis (p=0.01) and a slight increase of in the late stage apoptosis (p=0.13). PS-341 induced apoptosis also in bone marrow samples from two patients with classical and aplastic PNH as assessed by flow cytometry, based on CD59 and CD55 expression in the CD34+ population (Figure 1). GPI-AP-CD34+ deficient cells were more sensitive to PS-341 at the early stage of apoptosis (at 50 nM, 14% vs 4.6%), as well as late stage (at 200nM: 22% vs 7.2%). In conclusion, we demonstrate that GPI-AP-deficient cells are more susceptible to proteasome inhibition through the activation of the proapoptotic/terminal UPR, which leads to specific apoptosis in these cells. The sensitivity of PNH cells in vivo and in vitro to proteasome inhibitors might allow for the development of therapeutic strategies specifically directed to target one or several components of the UPR in PNH.Figure 1:Representative apoptosis assay in CD34+ cells according to GPI expression after 24 hours exposition to PS-341Figure 1:. Representative apoptosis assay in CD34+ cells according to GPI expression after 24 hours exposition to PS-341 Disclosures: No relevant conflicts of interest to declare.
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Galli, Monica, Paul Comfurius, Tiziano Barbui, Robert F. A. Zwaal i Edouard M. Bevers. "Anticoagulant Activity of β2-Glycoprotein I Is Potentiated by a Distinct Subgroup of Anticardiolipin Antibodies". Thrombosis and Haemostasis 68, nr 03 (1992): 297–300. http://dx.doi.org/10.1055/s-0038-1656368.
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SummaryPlasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study.Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma.These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of β2-glycoprotein I (β2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using β2-GPI deficient plasma, but could be restored upon addition of β2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of β2-GPI.Since β2-GPI binds to negatively-charged phospholipids and impedes the conversion of prothrombin by the factor Xa/Va enzyme complex (Nimpf et al., Biochim Biophys Acta 1986; 884: 142–9), comparison was made of the effect of aCL-type A and aCL-type B antibodies on the rate of thrombin formation in the presence and absence of β2-GPI. This was measured in a system containing highly purified coagulation factors Xa, Va and prothrombin and lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. No inhibition on the rate of thrombin formation was observed with both types of aCL antibodies when either β2-GPI or the lipid vesicles were omitted. Addition of β2-GPI to the prothrombinase assay in the presence of lipid vesicles causes a time-dependent inhibition which was not affected by the presence of aCL-type B or non-specific IgG. In contrast, the presence of aCL-type A antibodies dramatically increased the anticoagulant effect of β2-GPI. These data indicate that the anticoagulant activity of aCL-type A antibodies in plasma is mediated by β2-GPI.
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Lieberman, Michael M., Vivek R. Nerurkar, Haiyan Luo, Bruce Cropp, Ricardo Carrion, Melissa de la Garza, Beth-Ann Coller i in. "Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys". Clinical and Vaccine Immunology 16, nr 9 (wrzesień 2009): 1332–37. http://dx.doi.org/10.1128/cvi.00119-09.
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ABSTRACT The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 μg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 μg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-μg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.
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Chavakis, Triantafyllos, Sandip M. Kanse, Barbara Yutzy, H. Roger Lijnen i Klaus T. Preissner. "Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes". Blood 91, nr 7 (1.04.1998): 2305–12. http://dx.doi.org/10.1182/blood.v91.7.2305.
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Abstract Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK−), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.
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Chavakis, Triantafyllos, Sandip M. Kanse, Barbara Yutzy, H. Roger Lijnen i Klaus T. Preissner. "Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes". Blood 91, nr 7 (1.04.1998): 2305–12. http://dx.doi.org/10.1182/blood.v91.7.2305.2305_2305_2312.
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Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK−), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.
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Araten, David J., Ken Csehak, Leah Zamechek, Jeesun Park, Cynthia Liu, Sherif Ibrahim i Amitabha Mazumder. "Identification of Ex Vivo Myeloma Cells with the PNH Phenotype". Blood 120, nr 21 (16.11.2012): 3994. http://dx.doi.org/10.1182/blood.v120.21.3994.3994.
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Abstract Abstract 3994 Myeloma cells can harbor ∼ 35 mutations, whereas according to Loeb's model, if > 2 oncogenic mutations are required for the development of a malignancy, then an elevation in the mutation rate would be required for these mutations to occur in the same cell. Indeed, there is evidence that hypermutability in myeloma may be a result of abnormal homologous recombination or activation induced cytidine deaminase. An alternative model is that successive rounds of clonal selection in an expanding pre-malignant population could result in an accumulation of oncogenic mutations. Using our method for the detection of rare cells that have an acquired somatic mutation of the PIG-A gene, we have previously demonstrated hypermutability in some but not all lymphoma and myeloma cell lines, and we have recently shown that samples of ex vivo blasts from ∼ 50% of patients with ALL demonstrate hypermutability. Here we have hypothesized that genomic instability could be identified in myeloma. PIG-A encodes an enzyme that is necessary for the biosynthesis of glycosylphosphatidylinositol (GPI) and is particularly promising as a sentinel gene for spontaneous mutations because it is X-linked, and thus a single mutation can produce the mutant phenotype. PIG-A is mutated in Paroxysmal Nocturnal Hemoglobinuria (PNH), and it is known from this condition that a broad spectrum of inactivating mutations can produce the PNH phenotype, which is a loss of all GPI-linked surface proteins. PIG-A mutations do not affect transmembrane proteins and are growth-neutral under almost all circumstances. The PNH phenotype can be readily detected by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD48, CD55, and CD59), as well the FLAER reagent, which binds directly to GPI. In order to quantitate the frequency of myeloma cells with the PNH phenotype, we analyzed thawed ficolled samples from patients with a heavy burden of myeloma cells in the marrow. Cells were stained sequentially with FLAER-Alexa 488, then with a mixture of murine anti-CD48, anti-CD55, and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin, followed by a PE-conjugated antibody specific for a myeloma-specific transmembrane protein–either CD38 or CD138. Live myeloma cells were identified by forward/side scatter and propidium iodide exclusion and expression of CD38 or CD138. Using this approach, in previous studies we have seen that cells with the PNH phenotype have a low FITC/FLAER fluorescence, defined as < 4% of the level of the GPI (+) cell population, after gating for cells with at least 10% of the PE fluorescence of the GPI (+) population. For a negative control, we analyzed 2 non-malignant B-lymphoblastoid cell lines (BLCLs) from normal donors, and for a positive control, we analyzed the mantle cell lymphoma cell line HBL2A (in this case using CD45-PE to identify transmembrane proteins). The normal BLCLs demonstrated a frequency of PNH cells of 6.3 × 10−6 and 18.4 × 10−6, which is in the range that we have previously reported for BLCLs and granulocytes from normal individuals. These values are also similar to the frequency of spontaneously arising phenotypic variants in normal donors using other genes. In contrast, as we have previously reported, the mantle cell line demonstrated a markedly higher frequency of cells with the PNH phenotype– 1034 × 10−6. Of the involved marrow samples analyzed, there were at least 2 distinct groups. One group, representing 14 of the 20 samples (70%), demonstrated a mutant frequency that is comparable to non-malignant cell populations, with a median value of 9.5 × 10−6 (range 2.4 to 37 × 10−6). The remaining 6 samples (30%) demonstrated a markedly increased frequency of PNH cells, with a median value of 90 × 10−6 (range 73 to 11,763 × 10−6). Most of the samples analyzed were obtained from patients who had received prior therapy, but one of the samples demonstrating a very high frequency of PNH cells (1314 × 10−6) was derived from a patient who had not had prior therapy and was known to have had an abnormality of p53 based on FISH. This data demonstrates that an increase in inactivating mutations—as determined by this assay– is not essential for the development of myeloma, but it does seem to be a common feature of this condition. This flow-based assay could be applied at the time of diagnosis to facilitate investigations as to whether hypermutability correlates with outcome in patients with myeloma. Disclosures: No relevant conflicts of interest to declare.
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Conti, Fabrizio, Antonella Capozzi, Simona Truglia, Emanuela Lococo, Agostina Longo, Roberta Misasi, Cristiano Alessandri, Guido Valesini i Maurizio Sorice. "The Mosaic of “Seronegative” Antiphospholipid Syndrome". Journal of Immunology Research 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/389601.
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In the clinical practice it is possible to find patients with clinical signs suggestive of antiphospholipid syndrome (APS), who are persistently negative for the laboratory criteria of APS, that is, anti-cardiolipin antibodies (aCL), anti-β2-GPI antibodies and lupus anticoagulant. Therefore, it was proposed for these cases the term of seronegative APS (SN-APS). In order to detect autoantibodies with different methodological approaches, sera from 24 patients with SN-APS were analysed for anti-phospholipid antibodies using TLC immunostaining, for anti-vimentin/cardiolipin antibodies by enzyme-linked immunosorbent assay (ELISA), and for anti-annexin V and anti-prothrombin antibodies by ELISA and dot blot. Control groups of our study were 25 patients with APS, 18 with systemic lupus erythematosus (SLE), and 32 healthy controls. Results revealed that 13/24 (54.2%) SN-APS sera were positive for aCL (9 of whom were also positive for lysobisphosphatidic acid) by TLC immunostaining, 11/24 (45.8%) for anti-vimentin/cardiolipin antibodies, 3/24 (12.5%) for anti-prothrombin antibodies, and 1/24 (4.2%) for anti-annexin V antibodies. These findings suggest that in sera from patients with SN-APS, antibodies may be detected using “new” antigenic targets (mainly vimentin/cardiolipin) or methodological approaches different from traditional techniques (mainly TLC immunostaining). Thus, SN-APS represents a mosaic, in which antibodies against different antigenic targets may be detected.
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Araten, David J., Ken Csehak, Daniel Anscher i Leah Zamechek. "Quantitative Analysis of the Effects of MYC Overexpression on the Mutation Rate in Human Cells Using the PIG-A Gene". Blood 118, nr 21 (18.11.2011): 4406. http://dx.doi.org/10.1182/blood.v118.21.4406.4406.
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Abstract Abstract 4406 It has been hypothesized that the early acquisition of genomic instability is essential to account for the high number of mutations commonly identified in human cancers. Previous reports have demonstrated that MYC overexpression results in gene amplification. Given the importance of MYC in Burkitt’s lymphoma, we investigated the effects of MYC on spontaneous inactivating somatic mutations, using an assay that we have developed, based on the PIG-A gene. PIG-A is X-linked, thus a single mutation can disrupt its function. PIG-A encodes an enzyme required for the biosynthesis of glycosylphosphatidylinositol (GPI), which is required for the expression of a set of membrane proteins on the cell surface (e.g., CD48, CD52, CD55, and CD59). Expansion of PIG-A mutant stem cells occurs in PNH, and it is known from this disorder that a very broad spectrum of mutations can inactive PIG-A. Using PIG-A, we have previously determined the mutation rate (μ) in EBV transformed B-lymphoblastoid cell lines (BLCLs) from normal human donors, and we have demonstrated genomic instability in cell lines derived from hematologic malignancies. Here we have measured μ in P493, a cell line derived from normal human B cells, which overexpress MYC (normalized levels 17 × higher than BLCLs). P493 can also be induced to express lower levels of MYC, resulting in a lower growth rate, using tetracycline and estradiol. To measure μ, pre-existing mutants are first eliminated from the culture by staining the cells with an antibody specific for CD59 and sorting to collect the upper 50th percentile of the population. This purified GPI(+) population is then returned to culture for 3 weeks, with careful cell counts taken to estimate the number of cell divisions (d) occurring in vitro. After expansion, the frequency (f) of mutants arising in vitro is measured by first incubating with a mixture of mouse antibodies specific for CD48, CD52, CD55, and CD59, followed by a rabbit anti-mouse PE conjugate, followed by a FITC conjugated antibody specific for a transmembrane protein (CD45 or HLA-DR). Live cells are identified based on exclusion of propidium iodide and based on FITC fluorescence; GPI(−) cells are defined as having <4% of the mean PE fluorescence as the GPI(+) population. f is calculated as # GPI(−) cells / total gated live cells. The mutation rate is then calculated as μ = f /d. In initial experiments, 6 separate cultures of P493 were analyzed, demonstrating a median μ of 15 × 10−7 mutations per cell division (range 11 – 33 × 10−7). For comparison, the positive control, a hypermutable mantle cell lymphoma cell line (HBL2), demonstrated a μ of 674 × 10−7, and five BLCLs derived from normal donors demonstrated a median μ value of 37 × 10−7 (range 11 to 57 × 10−7). In parallel experiments, P493 was also grown in the presence of tetracycline and estradiol to reduce MYC: in 5 separate experiments, P493 growing in these conditions demonstrated a median μ of 6 × 10−7. However, in 2 subsequent experiments, we observed a higher μ under “lower MYC” conditions: 81 × 10−7 (SEM ±15 × 10−7) vs 13 × 10−7 (±2.2 × 10−7) and 65 × 10−7 (±6.2 × 10−7) vs 21 × 10−7(±3.3 × 10−7). We then isolated 6 clones of P493: for 5 clones, under “high MYC” conditions, μ was similar to the previous experiments (26 × 10−7, SEM ±3.4 × 10−7), and for 3 of these clones μ went up in the “lower MYC” conditions by 5 to 9 fold. For 2 of these clones, μ was similar in the “high MYC” and “lower MYC” conditions. One of the 6 clones, surprisingly, did not adopt a lower growth rate in the presence of tetracycline and estradiol and demonstrated a reversed pattern: μ was 70 × 10−7 under “high MYC” conditions and 16 × 10−7 under “lower MYC” conditions. We next analyzed 7 cell lines derived from Burkitt’s neoplasms which also overexpressed MYC (mean normalized levels ~ 3 × higher compared with normal BLCLs). Here we found a bimodal pattern, as we have previously described for primary acute lymphoblastic leukemia samples: for 4 of the cell lines μ was < 25 × 10−7, whereas 3 samples exhibited μ values comparable to the hypermutable positive control: 229 × 10−7, 562 × 10−7, and 1786 × 10−7 respectively. We conclude that overexpression of MYC is not sufficient to produce hypermutability in human B cells, based on this assay for gene-inactivating mutations. Hypermutability–as detected here–is a common feature of cell lines derived from Burkitt’s neoplasms but does not seem to be necessary for its development. Disclosures: No relevant conflicts of interest to declare.
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Araten, David J., Mignon L. Loh, Meenakshi Devidas, Andrew J. Carroll, Nyla A. Heerema, Stephen P. Hunger, Chris Amro i Leah Zamechek. "Leukemic Blasts With The PNH Phenotype: Correlation With Cytogenetics In ALL". Blood 122, nr 21 (15.11.2013): 2628. http://dx.doi.org/10.1182/blood.v122.21.2628.2628.
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Abstract It has been proposed that hypermutability is essential for the generation of malignancies that require more than a few oncogenic mutations and may also promote the emergence of drug-resistant clones by contributing to intratumoral diversity. However, most genes are not suitable as a reporter for quantitating the frequency of spontaneous somatic inactivating mutations. A notable exception is the PIG-A gene, which encodes an enzyme catalyzing an initial step in the biosynthesis of glycosylphosphatidylinositol (GPI), which links certain membrane proteins to the cell surface. Of note, the PIG-A-mutant phenotype can be readily demonstrated by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD55 and CD59), as well as the FLAER reagent (which binds to GPI directly), enabling the rapid screening of large populations for rare variants that are GPI-negative. It is known from the condition paroxysmal nocturnal hemoglobinuria that a very broad spectrum of PIG-A mutations can produce this “PNH phenotype”, and because the gene is X-linked, this requires only a single mutation. Apart from patients with PNH, there is evidence that PIG-A mutations are growth-neutral in most all other circumstances. We have previously demonstrated rare spontaneously arising cells with the PNH phenotype and genotype in blood cells of normal donors and in B-lymphoblastoid cell lines (BLCLs); similar populations can be demonstrated in mice, with marked increases after mutagen exposure. We have also shown that malignant cell lines often demonstrate an elevated frequency (f) of spontaneously arising cells with the PNH phenotype. Recently, we have reported similar findings in ex vivo leukemic blasts derived from patients with ALL: in samples from 10 of 19 patients, the median frequency of blasts with the PNH phenotype was low, with a median f value of 13 per million, which was similar to that of BLCLs from normal donors. The remaining 9 samples demonstrated an elevated f value (median 566 per million). Based on our preliminary subset analysis, along with published data demonstrating a higher mutation burden in leukemias with the ETV6-RUNX1 (TEL-AML1) translocation compared with hyperdiploid samples, we hypothesized that we would find a higher mutant frequency in the former group. Therefore, we have now analyzed in a blinded manner a new panel of blast cells derived from ficolled pre-treatment marrow samples from patients with B-lineage ALL obtained from the Children’s Oncology Group ALL Cell Bank. To determine f, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent and then a mixture of mouse anti-CD55 and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody (registering on FL1), and then PE-conjugated anti-CD45 antibody (registering on FL2). The blast population was identified based on forward and side scatter, expression of CD45, and exclusion of propidium iodide. GPI-negative cells were defined as expressing <4% of the mean level of FL1 fluorescence as the GPI-positive population, and >10% of its mean FL2 fluorescence. Overall, among 26 ALL samples, the f values spanned 5 orders of magnitude and had a median value of 49 per million. For the 8 ETV6-RUNX1 samples, the median f value was 2243 per million, compared with 18 per million for the 11 hyperdiploid samples (p <0.003). Among 7 samples with neither abnormality, the median f value was 60 per million. The one sample with a t(1;19) translocation that we analyzed demonstrated an f value of 330 per million. In comparison, the median f value among 11 BLCLs from normal donors was ∼ 5 per million, and f was ∼1850 per million for HBL2A, a mantle cell lymphoma line that exhibits hypermutability. Based on the data for BLCLs, we used a value of 50 per million as a cutoff: 7 of 8 ETV6-RUNX1 samples demonstrated hypermutabilty, compared with 2 of 11 hyperdiploid samples, and 4 of 7 samples with neither abnormality. These data demonstrate that hypermutability is frequently seen in ALL and is associated with, but not restricted to samples with the ETV6-RUNX1 fusion. These data also confirm that hypermutability—at least as detected by this assay, is not always a requirement for leukemogenesis. We predict that it will be possible to find associations between other cytogenetic subsets and hypermutability, which may correlate with the number of mutations required to produce the malignancy. Disclosures: No relevant conflicts of interest to declare.
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Mansy, Amr A., Nermeen N. Al-Gizawi, AbdEl-Latif Gaballah, Noha AbdEl El-Sawi i Wessam AL-Gendi. "The clinical value of measuring antiphospholipid antibodies in high risk primigravidae and nullipara, a case control study". International Journal of Reproduction, Contraception, Obstetrics and Gynecology 6, nr 12 (23.11.2017): 5198. http://dx.doi.org/10.18203/2320-1770.ijrcog20175227.
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Background: Antiphospholipid syndrome (APS) is an autoimmune thrombophilic condition that is marked by the presence in blood of antibodies that recognize and attack phospholipid-binding proteins, rather than phospholipid itself. The clinical manifestations of APS include vascular thrombosis and pregnancy complications, especially recurrent spontaneous miscarriages and, less frequently maternal thrombosis. Obstetric manifestations of APS are not restricted to fetal loss. Current APS criteria include preterm labour, oligohydramnios, neonatal complications as prematurity-estimated at 30-60% and more common in SLE patients, intrauterine growth restriction “IUGR”, fetal distress and rarely fetal or neonatal thrombosis, associated maternal obstetric complications as pre-eclampsia/eclampsia and HELLP syndrome, arterial or venous thrombosis and other aPL-related complications as placental insufficiency.Methods: The study included 90 primigravida and nullipara during their first trimester antenatal care visits, divided into 2 groups after signing a well-informed consent to declare their willing to participate in the study. All selected cases and control group were subjected to demographic data, thorough history taking, clinical examination focused on arterial blood pressure, body mass index, laboratory investigations fasting and random blood glucose level, serum analysis of anti-phospholipid anti bodies by enzyme linked immunosorbent assay (ELISA) including anticardiolipin antibodies (ACL), anti β2-glycoprotein I antibodies (Anti β2 GPI) and citrated sample for lupus anticoagulant test (LA).Results: We can conclude from the current study that aPLs namely lupus anticoagulant is significantly more common in high risk primigravidae having obesity, hypertension and diabetes mellitus than those without risk factors.Conclusions: aPLs antibodies as lupus anticoagulant is significantly more common in high risk primigravidae having obesity, hypertension and diabetes mellitus than those without risk factors.
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Miao, Li, i Qibin Lu. "Anzi Heji Downregulates DNMT1 to Improve Anticardiolipin Antibody (ACA)-Positive Abortion by Regulating JAK/STAT Pathway". Natural Product Communications 17, nr 7 (lipiec 2022): 1934578X2211128. http://dx.doi.org/10.1177/1934578x221112813.
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Anzi Heji (AZHJ) is a traditional Chinese medicine compound prepared for long-term treatment of Anticardiolipin Antibody (ACA)-positive abortion, with small side effects and definite curative effect. Abortion was reported to be related to DNMT1, a methylation transferase regulated by JAK2 pathway, so this study aimed to explore whether AZHJ treated ACA-positive abortion by regulating the DNMT1. Cell proliferation estimation employed Cell counting kit-8 (CCK-8) and flow cytometry. Human β2-glycoprotein I (GPI) was used as an inducer to establish ACA-positive mice model. Western blot was applied to examine the expressions of DNMT1, FOXP3, IL-6, and JAK/STAT3 pathway-related proteins. ACA titers and IL-6 levels in peripheral blood were tested by enzyme-linked immunosorbent assay (ELISA). Placental tissue damage was assessed by hematoxylin and eosin (H&E) staining. Based on the findings from experiments, AZHJ could significantly inhibit apoptosis and regulate the proliferation activity of HTR-8/SVneo cells. AZHJ treatment reduced the expression levels of DNMT1, FOXP3, IL-6, and JAK/STAT3 signaling pathways-related proteins in HTR-8/SVneo cells and maternal–fetal interface (uterine decidua and placenta), and the titer of serum ACA was also significantly decreased. In addition, AZHJ effectively alleviated placental tissue damage caused by ACA-positive abortion compared with model group. To sum up, AZHJ may play a therapeutic role by inhibiting DNMT1 activation through Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and then promoting FOXP3 expression in maternal–fetal interface of pregnant mice, thereby improving immune tolerance at the maternal–fetal interface, preventing and treating ACA-positive abortion.
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Sun, Jingjing, Xue Li, Haotian Zhou, Xiaoyun Liu, Jingjing Jia, Qizhi Xie, Sijia Peng, Xiaolin Sun, Qingwen Wang i Li Yi. "Anti-GAPDH Autoantibody Is Associated with Increased Disease Activity and Intracranial Pressure in Systemic Lupus Erythematosus". Journal of Immunology Research 2019 (31.03.2019): 1–9. http://dx.doi.org/10.1155/2019/7430780.
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Objective. Systemic lupus erythematosus (SLE) is an immune disease characterized by multiorgan involvement. Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most devastating complications of SLE, which lacks efficient diagnostic biomarkers. The recent studies on the anti-GAPDH autoantibodies suggested its potential pathogenic roles in NPSLE. However, the clinical relevance of the anti-GAPDH autoantibodies in patients with SLE is still elusive. In this study, we sought to determine the serum levels of the anti-GAPDH autoantibodies in patients with SLE to investigate the clinical significance of the anti-GAPDH autoantibodies in SLE. Methods. Concentrations of the glyceraldehyde 3-phosphate dehydrogenase autoantibodies (anti-GAPDH autoantibodies) in the serum of 130 SLE patients and 55 healthy individuals were determined by enzyme-linked immunosorbent assay (ELISA). Among the 130 SLE patients, 95 were SLE patients without neuropsychiatric symptoms and 35 had NPSLE. White blood cell (WBC) count, hemoglobin (HB), platelet count (PLT), IgG, IgA, IgM, anti-dsDNA, C3, C4, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), RF, anti-cardiolipin (Acl), ANA, AnuA, anti-SSA, anti-SSB, β2-GPI, urinalysis, and 24 h urine protein were measured by standard laboratory techniques. Systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index scores were evaluated accordingly. Results. The serum levels of the anti-GAPDH autoantibodies were significantly elevated in the SLE patients, especially in the patients with NPSLE (P=0.0011). Elevated serum anti-GAPDH was correlated with increased SLEDAI-2K (P=0.017), ESR, IgG, and IgM and associated with increased intracranial pressure and incidence of cerebrovascular lesions, but it was protective for seizure disorder incidence. Conclusions. Serum anti-GAPDH autoantibody was increased in both groups of SLE patients with or without neuropsychiatric symptoms and associated with disease severity. It could become an indicator of tissue damages in the brain for the future clinical practice.
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Hao, Ying, Jing Wang, Ningguo Feng i Anson W. Lowe. "Determination of Plasma Glycoprotein 2 Levels in Patients With Pancreatic Disease". Archives of Pathology & Laboratory Medicine 128, nr 6 (1.06.2004): 668–74. http://dx.doi.org/10.5858/2004-128-668-dopgli.
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Abstract Context.—Blood tests possessing higher diagnostic accuracy are needed for all the major pancreatic diseases. Glycoprotein 2 (GP2) is a protein that is specifically expressed by the pancreatic acinar cell and that has previously shown promise as a diagnostic marker in animal models of acute pancreatitis. Objective.—This study describes the development of an assay for GP2, followed by the determination of plasma GP2 levels in patients with acute pancreatitis, chronic pancreatitis, and pancreatic cancer. Design.—Rabbit polyclonal antisera and mouse monoclonal antibodies were generated against human GP2 and used to develop an enzyme-linked immunosorbent assay. The assay was tested in patients with an admitting diagnosis of pancreatic disease at 2 tertiary care facilities. The diagnosis of acute or chronic pancreatitis and pancreatic cancer was determined using previously established criteria that incorporated symptoms, radiology, pathology, and serology. Plasma GP2 levels were determined in 31 patients with acute pancreatitis, 16 patients with chronic pancreatitis, 36 patients with pancreatic cancer, and 143 control subjects without pancreatic disease. Amylase and lipase levels were also determined in patients with acute pancreatitis. Results.—The GP2 assay's sensitivity values were 0.94 for acute pancreatitis, 0.81 for chronic pancreatitis, and 0.58 for pancreatic cancer, which were greater than the 0.71 for acute pancreatitis and 0.43 for chronic pancreatitis (P = .02) observed for amylase. The lipase assay sensitivity for acute pancreatitis was 0.66. The accuracy of the GP2 assay was greater than that of the amylase or lipase assays for acute pancreatitis (GP2 vs lipase, P = .004; GP2 vs amylase, P = .003) when analyzed using receiver operator characteristic curves. When daily serial blood samples were obtained for 13 patients with acute pancreatitis, GP2 levels remained abnormally elevated for at least 1 day longer than the amylase or lipase levels. Conclusion.—The GP2 assay is a useful new marker for acute and chronic pancreatitis.
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Howes, N. K., M. I. Kovacs, D. Leisle, M. R. Dawood i W. Bushuk. "Screening of durum wheats for pasta-making quality with monoclonal antibodies for gliadin 45". Genome 32, nr 6 (1.12.1989): 1096–99. http://dx.doi.org/10.1139/g89-560.
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Monoclonal antibodies specific for gliadin band 45 (gli 45) of common wheat cultivar 'Marquis' were treated against durum wheat gliadins using an enzyme-linked immunosorbent assay (ELISA). In a test of 15 durum cultivars, high ELISA values were associated with gli 45, high gluten strength, and high sodium dodecyl sulfate (SDS) sedimentation volume; low ELISA values were associated with gli 42, low gluten strength, and low SDS-sedimentation volume. Analysis of 120 F5 lines from the backcrosses Ward/Vic//Vic and Ward/Vic//Ward confirmed that a high reaction to ELISA was statistically correlated with the presence of gli 45, with high gluten strength, and with high SDS-sedimentation volume. It was concluded that monoclonal antibodies specific for gli 45 have potential as a test for rapid screening of durum wheat breeding populations for desirable pasta-making quality.Key words: monoclonal antibodies, gliadin, enzyme-linked immunosorbent assay, durum wheat.
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Dai, Ying-Chun, Ming Xia, Qiong Huang, Ming Tan, Lin Qin, Ya-Li Zhuang, Yan Long, Jian-Dong Li, Xi Jiang i Xu-Fu Zhang. "Characterization of Antigenic Relatedness between GII.4 and GII.17 Noroviruses by Use of Serum Samples from Norovirus-Infected Patients". Journal of Clinical Microbiology 55, nr 12 (13.09.2017): 3366–73. http://dx.doi.org/10.1128/jcm.00865-17.
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ABSTRACTA novel GII.17 norovirus variant caused major gastroenteritis epidemics in China in 2014 to 2016. To explore the host immune factors in selection of the emergence of this new variant, we characterized its antigenic relatedness with the GII.4 noroviruses that have dominated in China for decades. Through an enzyme-linked immunosorbent assay (ELISA) and a histo-blood group antigen (HBGA) blocking assay using sera from GII.4 and the GII.17 variant-infected patients, respectively, we observed limited cross-immune reactivity by the ELISA but little reactivity by the HBGA blocking assay between GII.4 norovirus and the new GII.17 variant. Our data suggest that, among other possible factors, GII.4-specific herd immunity had little role in the emergence of the new GII.17 variant. Thus, GII.17 may be an important active antigenic type or immunotype that needs to be considered for future vaccine strategies against human noroviruses.
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Lamhoujeb, Safaa, Hugues Charest, Ismail Fliss, Solange Ngazoa i Julie Jean. "Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples". Canadian Journal of Microbiology 55, nr 12 (grudzień 2009): 1375–80. http://dx.doi.org/10.1139/w09-105.
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To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.
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Mesquita, João Rodrigo, i Maria São José Nascimento. "Norovirus GII.4 antibodies in the Portuguese population". Journal of Infection in Developing Countries 8, nr 09 (12.09.2014): 1201–4. http://dx.doi.org/10.3855/jidc.4616.
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Introduction: Norovirus GII.4 is the leading cause of outbreaks of acute and sporadic acute gastroenteritis worldwide. Information on the prevalence of norovirus in Portugal is scarce or null. Methodology: We used a GII.4 norovirus virus-like particle-based enzyme immune assay to measure the seropositivity rate of GII.4 norovirus. Results: A total of 342 (70%) out of 473 serum samples tested positive for anti-GII.4 norovirus IgG. No statistically significant differences were found between regions, sex and age groups. Conclusion: Norovirus GII.4 infections are frequent in Portugal.
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Ercan-Fang, Nacide, Miriam R. Taylor, Judith L. Treadway, Carolyn B. Levy, Paul E. Genereux, E. Michael Gibbs, Virginia L. Rath, Younggil Kwon, Mary C. Gannon i Frank Q. Nuttall. "Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors". American Journal of Physiology-Endocrinology and Metabolism 289, nr 3 (wrzesień 2005): E366—E372. http://dx.doi.org/10.1152/ajpendo.00264.2004.
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Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10–30 μM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.
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Cuschieri, K., D. T. Geraets, C. Moore, W. Quint, E. Duvall i M. Arbyn. "Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework". Journal of Clinical Microbiology 53, nr 10 (5.08.2015): 3272–79. http://dx.doi.org/10.1128/jcm.01366-15.
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As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n= 1,000), enriched with cytologically abnormal samples (n= 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (Pfor trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05;P< 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00;P= 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.
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Liang, Yu, Wei Bu Wang, Jing Zhang, Jun Wei Hou, Fang Tang, Xue Feng Zhang, Li Fang Du, Ji Guo Su i Qi Ming Li. "Evolution of the interactions between GII.4 noroviruses and histo-blood group antigens: Insights from experimental and computational studies". PLOS Pathogens 17, nr 7 (12.07.2021): e1009745. http://dx.doi.org/10.1371/journal.ppat.1009745.
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Norovirus (NoV) is the major pathogen causing the outbreaks of the viral gastroenteritis across the world. Among the various genotypes of NoV, GII.4 is the most predominant over the past decades. GII.4 NoVs interact with the histo-blood group antigens (HBGAs) to invade the host cell, and it is believed that the receptor HBGAs may play important roles in selecting the predominate variants by the nature during the evolution of GII.4 NoVs. However, the evolution-induced changes in the HBGA-binding affinity for the GII.4 NoV variants and the mechanism behind the evolution of the NoV-HBGA interactions remain elusive. In the present work, the virus-like particles (VLPs) of the representative GII.4 NoV stains epidemic in the past decades were expressed by using the Hansenula polymorpha yeast expression platform constructed by our laboratory, and then the enzyme linked immunosorbent assay (ELISA)-based HBGA-binding assays as well as the molecular dynamics (MD) simulations combined with the molecular mechanics/generalized born surface area (MMGBSA) calculations were performed to investigate the interactions between various GII.4 strains and different types of HBGAs. The HBGA-binding assays show that for all the studied types of HBGAs, the evolution of GII.4 NoVs results in the increased NoV-HBGA binding affinities, where the early epidemic strains have the lower binding activity and the newly epidemic strains exhibit relative stronger binding intensity. Based on the MD simulation and MMGBSA calculation results, a physical mechanism that accounts for the increased HBGA-binding affinity was proposed. The evolution-involved residue mutations cause the conformational rearrangements of loop-2 (residues 390–396), which result in the narrowing of the receptor-binding pocket and thus tighten the binding of the receptor HBGAs. Our experimental and computational studies are helpful for better understanding the mechanism behind the evolution-induced increasing of HBGA-binding affinity, which may provide useful information for the drug and vaccine designs against GII.4 NoVs.
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Yang, Yang, Ming Xia, Ming Tan, Pengwei Huang, Weiming Zhong, Xiao Li Pang, Bonita E. Lee, Jarek Meller, Tao Wang i Xi Jiang. "Genetic and Phenotypic Characterization of GII-4 Noroviruses That Circulated during 1987 to 2008". Journal of Virology 84, nr 18 (30.06.2010): 9595–607. http://dx.doi.org/10.1128/jvi.02614-09.
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ABSTRACT The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.
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Mazáň, Marián, Enrico Ragni, Laura Popolo i Vladimír Farkaš. "Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay". Biochemical Journal 438, nr 2 (12.08.2011): 275–82. http://dx.doi.org/10.1042/bj20110405.
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BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C3(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR–LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.
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Atmar, Robert L., David I. Bernstein, G. Marshall Lyon, John J. Treanor, Mohamed S. Al-Ibrahim, David Y. Graham, Jan Vinjé i in. "Serological Correlates of Protection against a GII.4 Norovirus". Clinical and Vaccine Immunology 22, nr 8 (3.06.2015): 923–29. http://dx.doi.org/10.1128/cvi.00196-15.
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ABSTRACTNoroviruses are the leading cause of acute gastroenteritis worldwide, and norovirus vaccine prevention strategies are under evaluation. The immunogenicity of two doses of bivalent genogroup 1 genotype 1 (GI.1)/GII.4 (50 μg of virus-like particles [VLPs] of each strain adjuvanted with aluminum hydroxide and 3-O-desacyl-4′monophosphoryl lipid A [MPL]) norovirus vaccine administered to healthy adults in a phase 1/2 double-blind placebo-controlled trial was determined using virus-specific serum total antibody enzyme-linked immunosorbent assay (ELISA), IgG, IgA, and histoblood group antigen (HBGA)-blocking assays. Trial participants subsequently received an oral live virus challenge with a GII.4 strain, and the vaccine efficacy results were reported previously (D. I. Bernstein et al., J Infect Dis 211:870–878, 2014, doi:10.1093/infdis/jiu497). This report assesses the impact of prechallenge serum antibody levels on infection and illness outcomes. Serum antibody responses were observed in vaccine recipients by all antibody assays, with first-dose seroresponse frequencies ranging from 88 to 100% for the GI.1 antigen and from 69 to 84% for the GII.4 antigen. There was little increase in antibody levels after the second vaccine dose. Among the subjects receiving the placebo, higher prechallenge serum anti-GII.4 HBGA-blocking and IgA antibody levels, but not IgG or total antibody levels, were associated with a lower frequency of virus infection and associated illness. Notably, some placebo subjects without measurable serum antibody levels prechallenge did not become infected after norovirus challenge. In vaccinees, anti-GII.4 HBGA-blocking antibody levels of >1:500 were associated with a lower frequency of moderate-to-severe vomiting or diarrheal illness. In this study, prechallenge serum HBGA antibody titers correlated with protection in subjects receiving the placebo; however, other factors may impact the likelihood of infection and illness after virus exposure. (This study is registered at ClinicalTrials.gov under registration number NCT1609257.)
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Sobarzo, Ariel, Eddie Perelman, Allison Groseth, Olga Dolnik, Stephan Becker, Julius Julian Lutwama, John M. Dye, Victoria Yavelsky, Leslie Lobel i Robert S. Marks. "Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay". Clinical and Vaccine Immunology 19, nr 11 (19.09.2012): 1844–52. http://dx.doi.org/10.1128/cvi.00363-12.
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ABSTRACTEbolavirus, a member of the familyFiloviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP1-294and GP1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.
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Pajuaba, Ana C. A. M., Deise A. O. Silva i José R. Mineo. "Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows". Clinical and Vaccine Immunology 17, nr 4 (10.02.2010): 588–95. http://dx.doi.org/10.1128/cvi.00444-09.
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ABSTRACT This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle.
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Wojewódzka, Maria, Marcin Kruszewski, Iwona Buraczewska, Weizheng Xu, Edmond Massuda, Jie Zhang i Irena Szumiel. "Sirtuin inhibition increases the rate of non-homologous end-joining of DNA double strand breaks." Acta Biochimica Polonica 54, nr 1 (20.02.2007): 63–69. http://dx.doi.org/10.18388/abp.2007_3270.
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Sirtuins (type III histone deacetylases) are an important member of a group of enzymes that modify chromatin conformation. We investigated the role of sirtuin inhibitor, GPI 19015, in double strand break (DSB) repair in CHO-K1 wt and xrs-6 mutant cells. The latter is defective in DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end-joining (D-NHEJ). DSB were estimated by the neutral comet assay and histone gammaH2AX foci formation. We observed a weaker effect of GPI 19015 treatment on the repair kinetics in CHO wt cells than in xrs6. In the latter cells the increase in DNA repair rate was most pronounced in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in the number of histone gammaH2AX foci was faster in xrs6 than in CHO-K1 cells. The altered repair rate did not affect survival of X-irradiated cells. Since in G1 xrs6 cells DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ, to a greater extent than the other DSB repair system, D-NHEJ.
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Saito, Daisuke, Yuko Nakagawa, Takashi Sato, Ayako Fukunaka, Ofejiro Blessing Pereye, Nobuhiro Maruyama, Hirotaka Watada i Yoshio Fujitani. "Establishment of an enzyme-linked immunosorbent assay for mouse pancreatic polypeptide clarifies the regulatory mechanism of its secretion from pancreatic γ cells". PLOS ONE 17, nr 8 (17.08.2022): e0269958. http://dx.doi.org/10.1371/journal.pone.0269958.
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Pancreatic polypeptide (PP), secreted from γ cells of the islets of Langerhans, is a 36 amino-acid peptide encoded by the Ppy gene. Although previous studies have reported that PP causes a decrease in appetite, the molecular mechanism that regulates PP secretion has not been fully elucidated. Lack of understanding of the regulatory mechanism of PP secretion may be partially owing to the lack of assay systems that can specifically detect PP. We recently developed the mouse monoclonal antibody 23-2D3 that specifically recognizes PP. In the present study, we developed a sandwich enzyme-linked immunosorbent assay for the measurement of mouse PP, and directly monitored intracellular Ca2+ concentrations in Ppy-expressing cells from a newly developed reporter mouse. Using these systems, we identified agonists, such as carbachol and glucose-dependent insulinotropic polypeptide (GIP), which stimulate PP secretion. We further demonstrated that, unlike the case of GIP-induced insulin secretion from β cells, there is a unique mechanism by which PP secretion is triggered by an increase in intracellular Ca2+ concentrations via voltage-dependent calcium channels even in low-glucose conditions.
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Roggenbuck, Dirk, Alexander Goihl, Katja Hanack, Pamela Holzlöhner, Christian Hentschel, Miklos Veiczi, Peter Schierack, Dirk Reinhold i Hans-Ulrich Schulz. "Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2". Clinical Chemistry and Laboratory Medicine (CCLM) 55, nr 6 (1.06.2017): 854–64. http://dx.doi.org/10.1515/cclm-2016-0797.
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Abstract Background: Glycoprotein 2 (GP2), the pancreatic major zymogen granule membrane glycoprotein, was reported to be elevated in acute pancreatitis in animal models. Methods: Enzyme-linked immunosorbent assays (ELISAs) were developed to evaluate human glycoprotein 2 isoform alpha (GP2a) and total GP2 (GP2t) as specific markers for acute pancreatitis in sera of 153 patients with acute pancreatitis, 26 with chronic pancreatitis, 125 with pancreatic neoplasms, 324 with non-pancreatic neoplasms, 109 patients with liver/biliary disease, 67 with gastrointestinal disease, and 101 healthy subjects. GP2a and GP2t levels were correlated with procalcitonin and C-reactive protein in 152 and 146 follow-up samples of acute pancreatitis patients, respectively. Results: The GP2a ELISA revealed a significantly higher assay accuracy in contrast to the GP2t assay (sensitivity ≤3 disease days: 91.7%, specificity: 96.7%, positive likelihood ratio [LR+]: 24.6, LR–: 0.09). GP2a and GP2t levels as well as prevalences were significantly elevated in early acute pancreatitis (≤3 disease days) compared to all control cohorts (p<0.05, respectively). GP2a and GP2t levels were significantly higher in patients with severe acute pancreatitis at admission compared with mild cases (p<0.05, respectively). Odds ratio for GP2a regarding mild vs. severe acute pancreatitis with lethal outcome was 7.8 on admission (p=0.0222). GP2a and GP2t levels were significantly correlated with procalcitonin [Spearman’s rank coefficient of correlation (ρ)=0.21, 0.26; p=0.0110, 0.0012; respectively] and C-reactive protein (ρ=0.37, 0.40; p<0.0001; respectively). Conclusions: Serum GP2a is a specific marker of acute pancreatitis and analysis of GP2a can aid in the differential diagnosis of acute upper abdominal pain and prognosis of severe acute pancreatitis.
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Sakamaki, Nozomi, Yoshiyuki Ohiro, Mitsuki Ito, Mitsuru Makinodan, Tsubasa Ohta, Wataru Suzuki, Susumu Takayasu i Harufumi Tsuge. "Bioluminescent Enzyme Immunoassay for the Detection of Norovirus Capsid Antigen". Clinical and Vaccine Immunology 19, nr 12 (17.10.2012): 1949–54. http://dx.doi.org/10.1128/cvi.00427-12.
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ABSTRACTAn ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y= 0.66x −3.21,r= 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 105to 106copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis.
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Ibrahim, Nihal Abdalla, Saba Kaleem, Abida Kalsoom Khan i Ghulam Murtaza. "Development and butyrylcholinesterase/monoamine oxidase inhibition potential of PVA-Berberis lycium nanofibers". Green Processing and Synthesis 11, nr 1 (1.01.2022): 229–37. http://dx.doi.org/10.1515/gps-2022-0017.
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Abstract The aim of this study was to evaluate the potential inhibitory effect of montmorillonite (MMT)-reinforced, glutaraldehyde-crosslinked PVA (polyvinyl alcohol) nanofibers loaded with root extract of Berberis lycium on monoamine oxidase A and B (MAO A and B) and butyrylcholinesterase (BChE) by using slightly modified Ellman’s test and Amplex Red monoamine oxidase assay, respectively. Enzyme inhibition studies of extract-loaded nanofibers showed significant inhibitory potential against MAO A, B, and BChE. There was an increase in enzyme inhibition with an increased extract concentration loaded to nanofibers. The fibers were characterized by TGA (thermal gravimetric analysis), SEM (scanning electron microscopy), XRD (X-ray diffractometry), and FTIR (Fourier-transform infra-red) spectroscopy to investigate thermal stability, morphology, structural changes, and functional groups in the nanofibers, respectively. SEM results of fabricated nanofibers reflected the beadless and smooth morphology of nanofibers with the porous structure. The contact angle measurements of fabricated nanofibers showed suitable hydrophilicity of nanofibers. The nanofibers loaded with the root extract of Berberis lycium have been found to be potent inhibitors of MAO A, B, and the BChE enzyme.
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Gao, Xiang, Malak A. Esseili, Zhongyan Lu, Linda J. Saif i Qiuhong Wang. "Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus". Applied and Environmental Microbiology 82, nr 10 (11.03.2016): 2966–74. http://dx.doi.org/10.1128/aem.04096-15.
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ABSTRACTHuman norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall.IMPORTANCESalad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination.
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Shirato, Haruko, Satoko Ogawa, Hiromi Ito, Takashi Sato, Akihiko Kameyama, Hisashi Narimatsu, Zheng Xiaofan i in. "Noroviruses Distinguish between Type 1 and Type 2 Histo-Blood Group Antigens for Binding". Journal of Virology 82, nr 21 (13.08.2008): 10756–67. http://dx.doi.org/10.1128/jvi.00802-08.
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ABSTRACT Norovirus (NoV) is a causative agent of acute gastroenteritis. NoV binds to histo-blood group antigens (HBGAs), namely, ABH antigens and Lewis (Le) antigens, in which type 1 and type 2 carbohydrate core structures constitute antigenically distinct variants. Norwalk virus, the prototype strain of norovirus, binds to the gastroduodenal junction, and this binding is correlated with the presence of H type 1 antigen but not with that of H type 2 antigen (S. Marionneau, N. Ruvoen, B. Le Moullac-Vaidye, M. Clement, A. Cailleau-Thomas, G. Ruiz-Palacois, P. Huang, X. Jiang, and J. Le Pendu, Gastroenterology 122:1967-1977, 2002). It has been unknown whether NoV distinguishes between the type 1 and type 2 chains of A and B antigens. In this study, we synthesized A type 1, A type 2, B type 1, and B type 2 pentasaccharides in vitro and examined the function of the core structures in the binding between NoV virus-like particles (VLPs) and HBGAs. The attachment of five genogroup I (GI) VLPs from 5 genotypes and 11 GII VLPs from 8 genotypes, GI/1, GI/2, GI/3, GI/4, GI/8, GII/1, GII/3, GII/4, GII/5, GII/6, GII/7, GII/12, and GII/14, to ABH and Le HBGAs was analyzed by enzyme-linked immunosorbent assay-based binding assays and Biacore analyses. GI/1, GI/2, GI/3, GI/4, GI/8, and GII/4 VLPs were more efficiently bound to A type 2 than A type 1, and GI/8 and GII/4 VLPs were more efficiently bound to B type 2 than B type 1, indicating that NoV VLPs distinguish between type 1 and type 2 carbohydrates. The dissociation of GII/4 VLPs from B type 1 was slower than that from B type 2 in the Biacore experiments; moreover, the binding to B type 1 was stronger than that to B type 2 in the ELISA experiments. These results indicated that the type 1 carbohydrates bind more tightly to NoV VLPs than the type 2 carbohydrates. This property may afford NoV tissue specificity. GII/4 is known to be a global epidemic genotype and binds to more HBGAs than other genotypes. This characteristic may be linked with the worldwide transmission of GII/4 strains. GI/2, GI/3, GI/4, GI/8, GII/4, and GII/7 VLPs bound to Lea expressed by nonsecretors, suggesting that NoV can infect individuals regardless of secretor phenotype. Overall, our results indicated that HBGAs are important factors in determining tissue specificity and the risk of transmission.
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Xu, Jiaqi, Junjie Yang, Yu Jiang, Mianbin Wu, Sheng Yang i Lirong Yang. "A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production". Acta Biochimica et Biophysica Sinica 53, nr 9 (25.06.2021): 1124–33. http://dx.doi.org/10.1093/abbs/gmab089.
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Abstract Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) β′ subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens’s innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI–GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.
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Collet, Laetitia, Corinne Vander Wauven, Yamina Oudjama, Moreno Galleni i Raphael Dutoit. "Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis". Acta Crystallographica Section D Structural Biology 77, nr 2 (26.01.2021): 205–16. http://dx.doi.org/10.1107/s2059798320015557.
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The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.
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Nimonkar, Amitabh V., Stephen Weldon, Kevin Godbout, Darrell Panza, Susan Hanrahan, Rose Cubbon, Fangmin Xu, John W. Trauger, Jiaping Gao i Andrei Voznesensky. "A lipoprotein lipase–GPI-anchored high-density lipoprotein–binding protein 1 fusion lowers triglycerides in mice: Implications for managing familial chylomicronemia syndrome". Journal of Biological Chemistry 295, nr 10 (23.10.2019): 2900–2912. http://dx.doi.org/10.1074/jbc.ra119.011079.
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Lipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol-anchored high-density lipoprotein–binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex and its derivative, the LPL–GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3 (ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL–GPIHBP1 fusion protein exhibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL–GPIHBP1 fusion protein has potential for use as a therapeutic for managing FCS.
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Crawford, Sue E., Nadim Ajami, Tracy Dewese Parker, Noritoshi Kitamoto, Katsuro Natori, Naokazu Takeda, Tomoyuki Tanaka, Baijun Kou, Robert L. Atmar i Mary K. Estes. "Mapping Broadly Reactive Norovirus Genogroup I and II Monoclonal Antibodies". Clinical and Vaccine Immunology 22, nr 2 (26.11.2014): 168–77. http://dx.doi.org/10.1128/cvi.00520-14.
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ABSTRACTNoroviruses are responsible for most acute nonbacterial epidemic outbreaks of gastroenteritis worldwide. To develop cross-reactive monoclonal antibodies (MAbs) for rapid identification of genogroup I and II (GI and GII) noroviruses (NoVs) in field specimens, mice were immunized with baculovirus-expressed recombinant virus-like particles (VLPs) corresponding to NoVs. Nine MAbs against the capsid protein were identified that detected both GI and GII NoV VLPs. These MAbs were tested in competition enzyme-linked immunosorbent assays (ELISAs) to identify common epitope reactivities to GI and GII VLPs. Patterns of competitive reactivity placed these MAbs into two epitope groups (groups 1 and 2). Epitopes for MAbs NV23 and NS22 (group 1) and MAb F120 (group 2) were mapped to a continuous region in the C-terminal P1 subdomain of the capsid protein. This domain is within regions previously defined to contain cross-reactive epitopes in GI and GII viruses, suggesting that common epitopes are clustered within the P1 domain of the capsid protein. Further characterization in an accompanying paper (B. Kou et al., Clin Vaccine Immunol 22:160–167, 2015,http://dx.doi.org/10.1128/CVI.00519-14) revealed that MAb NV23 (epitope group 1) is able to detect GI and GII viruses in stool. Inclusion of the GI and GII cross-reactive MAb NV23 in antigen detection assays may facilitate the identification of GI and GII human noroviruses in stool samples as causative agents of outbreaks and sporadic cases of gastroenteritis worldwide.
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Cuschieri, Kate, Daan Geraets, Jack Cuzick, Louise Cadman, Catherine Moore, Davy Vanden Broeck, Elisaveta Padalko, Wim Quint i Marc Arbyn. "Performance of a Cartridge-Based Assay for Detection of Clinically Significant Human Papillomavirus (HPV) Infection: Lessons from VALGENT (Validation of HPV Genotyping Tests)". Journal of Clinical Microbiology 54, nr 9 (6.07.2016): 2337–42. http://dx.doi.org/10.1128/jcm.00897-16.
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The Validation of Human Papillomavirus (HPV) Genotyping Tests (VALGENT) studies offer an opportunity to clinically validate HPV assays for use in primary screening for cervical cancer and also provide a framework for the comparison of analytical and type-specific performance. Through VALGENT, we assessed the performance of the cartridge-based Xpert HPV assay (Xpert HPV), which detects 14 high-risk (HR) types and resolves HPV16 and HPV18/45. Samples from women attending the United Kingdom cervical screening program enriched with cytologically abnormal samples were collated. All had been previously tested by a clinically validated standard comparator test (SCT), the GP5+/6+ enzyme immunoassay (EIA). The clinical sensitivity and specificity of the Xpert HPV for the detection of cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and CIN3+ relative to those of the SCT were assessed as were the inter- and intralaboratory reproducibilities according to international criteria for test validation. Type concordance for HPV16 and HPV18/45 between the Xpert HPV and the SCT was also analyzed. The Xpert HPV detected 94% of CIN2+ and 98% of CIN3+ lesions among all screened women and 90% of CIN2+ and 96% of CIN3+ lesions in women 30 years and older. The specificity for CIN1 or less (≤CIN1) was 83% (95% confidence interval [CI], 80 to 85%) in all women and 88% (95% CI, 86 to 91%) in women 30 years and older. Inter- and intralaboratory agreements for the Xpert HPV were 98% and 97%, respectively. The kappa agreements for HPV16 and HPV18/45 between the clinically validated reference test (GP5+/6+ LMNX) and the Xpert HPV were 0.92 and 0.91, respectively. The clinical performance and reproducibility of the Xpert HPV are comparable to those of well-established HPV assays and fulfill the criteria for use in primary cervical cancer screening.
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Li, Dan, Leen Baert, Dongsheng Zhang, Ming Xia, Weiming Zhong, Els Van Coillie, Xi Jiang i Mieke Uyttendaele. "Effect of Grape Seed Extract on Human Norovirus GII.4 and Murine Norovirus 1 in Viral Suspensions, on Stainless Steel Discs, and in Lettuce Wash Water". Applied and Environmental Microbiology 78, nr 21 (17.08.2012): 7572–78. http://dx.doi.org/10.1128/aem.01987-12.
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ABSTRACTThe anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.