Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The latent viral genome, harbored indefinitely, threatens reactivation from its remote location. Although polymerase chain reaction (PCR) has detected the organs responsible for latency, it is not known whether latent cytomegalovirus (CMV) infection is maintained within organ-specific cells or ubiquitous elements such as macrophages, endothelial cells, or perhaps others. PCR lacks correlation with tissue structure. However, PCR-based in situ hybridization maintains cellular architecture while allowing the identification of the latently infected cells. Murine CMV (MCMV) nucleic acid sequences in organs of latently infected Balb/C mice were amplified by PCR incorporating digoxigenin-11-dUTP, holding the product DNA in situ (appropriate controls analyzed in parallel). Product DNA was then hybridized in situ with a biotinylated oligonucleotide probe for detection via streptavidin-alkaline phosphatase and light microscopy. Immunohistochemistry verified the positive cell types. Using this technique, we have shown directly in multiple organs of latently infected Balb/C mice including kidney (5/5), liver (5/5), and spleen (5/5) that the endothelial cell and/or T-lymphocyte harbor latent MCMV, whereas in uninfected animals, MCMV DNA was not detected. PCR-based in situ hybridization allows detection of the specific cell(s) harboring latent MCMV DNA while allowing conservation of cellular architecture.
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PMID:Direct evidence using in situ polymerase chain reaction that the endothelial cell and T-lymphocyte harbor latent murine cytomegalovirus. 866 44

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.
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PMID:An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies. 873

The FLAG peptide, AspTyrLysAspAspAspAspLys, has been used as an epitope tag in a variety of cell types. The modification of the cytomegalovirus (CMV) promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described. As a functional test, the bacterial alkaline phosphatase gene was cloned into both vectors, and anti-FLAG monoclonal antibodies were used for detection of FLAG epitope-tagged bacterial alkaline phosphatase in mammalian cells. In addition, secreted bacterial alkaline phosphatase was purified from the extracellular medium by anti-FLAG affinity chromatography.
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PMID:Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells. 877 Apr 18

A 24 base pair oligonucleotide probe directly conjugated to alkaline phosphatase has been used to detect immobilised amplicons derived from a cytomegalovirus specific polymerase chain reaction (PCR). The sensitivity of detection using a highly amplified alkaline phosphatase detection system was four genome equivalents and was comparable to the limit of detection using agarose gel methods. The mean optical density at 492 nm of samples not known to contain cytomegalovirus DNA was 0.085 +/- 0.006 and was well separated from the optical density generated from four genome equivalents (absorption at 492 nm: 0.132). The assay was used to identify the presence of cytomegalovirus in blood DNA extracts from immunocompromised patients in whom conventional ethidium bromide stained agarose gel electrophoresis revealed the presence of multiple amplicons. Samples yielding an uninterpretable result at both neat and diluted 1 in 20 in the PCR gave rise to the highest proportion of positive results (68%) whilst samples that produced uninterpretable results neat but were negative at 1 in 20 and vice versa gave positive rates of 33.6 and 21.7%, respectively. The use of this assay for identifying cytomegalovirus specific PCR products in problematic samples is discussed.
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PMID:Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase. 888 47

Identification of human tissue contaminated by cytomegalovirus is currently carried out by PCR amplification followed by measurement of the amplicons. Three luminescent detection systems undertaken after sandwich hybridization of the amplicons were compared. The sandwich hybridization takes place between a covalent linked capture probe, bound onto a plastic 96-well plate, and a biotinylated or digoxigenin-labeled detection probe. The three non-isotopic luminescent detection systems need either streptavidin-conjugated peroxydase or streptavidin-conjugated pyruvate kinase or antibodies conjugated with alkaline phosphatase. Detection of the enzymes was carried out by measurement of light emission in the presence of, respectively, luminol for peroxidase or dioxethane for alkaline phosphatase. The kinase assay was carried out not only in the presence of its substrates, ADP and phospho-enol pyruvate, but also of luciferase, which converts the produced ATP into light. The method was found to be sensitive, with the luciferase bioluminescent assay with the production of a long lasting signal. Amplicons from eight clinical samples were detected by this combination of sandwich hybridization and the three luminescent assays. The results were comparable with nested PCR for the identification of positive samples. The same correlation was obtained with 45 clinical samples using only the pyruvate kinase detection system. The high performance of these assays is given by the specificity of the sandwich hybridization combined with the sensitivity of the luminescent detection systems.
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PMID:Comparison of three luminescent assays combined with a sandwich hybridization for the measurement of PCR-amplified human cytomegalovirus DNA. 922 Mar 97

The plaques or foci of certain viruses due to their small size have to be counted microscopically, e.g., human cytomegalovirus (HCMV). The focus luminescence assay (FLA) described below generates macroscopic images as a result of the magnification due to scattered emitted light, and provides a hard copy using autoradiography or video imaging. Foci are detected according to an immunohistochemical protocol with horseradish peroxidase or alkaline phosphatase antibody conjugates which convert substrate into a luminescent product. Detection of varicella zoster virus (VZV) foci developed with a specific substrate-enhancer combination was so sensitive that 20-times lower primary antibody concentrations were effective than those required for conventional immunohistochemical staining. This method for HCMV and VZV may allow quantitative infectivity and focus reduction assays for viruses which produce little or no CPE.
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PMID:Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection. 925 42

It's well known that patients was acquired immunodeficiency syndrome (AIDS) can develop various kinds of hepatobiliopancreatic diseases, for causes related to AIDS and for causes not related to HIV infection. The authors describe a case to their attention due to a suspected acute pancreatitis. The patient presented with abdominal pain, increased serum alkaline phosphatase and amylase levels. Serological test and stool concentration didn't show any opportunistic infection (Cytomegalovirus, Cryptosporidium). Abdominal ultrasonography showed enlargement of the head of the pancreas, gallbladder with biliary sludge, and a little dilatation of the biliary tree. The patient didn't feel better despite the medical treatment, so considering the probability of the migration of calculus, the patient underwent cholecystectomy. After the operation the patient felt better quickly. This case confirms the presence in HIV patients of pancreatitis for causes unrelated to AIDS like cholelithiasis as we showed, alcoholism, hypercalcemia, and the importance of an opportune surgical treatment that was resolutive.
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PMID:[Acute pancreatitis and AIDS]. 932 71

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.
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PMID:Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo. 937 79

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.
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PMID:Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization. 942 39

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH4Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.
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PMID:Rapid cytomegalovirus pp65 antigenemia assay by direct erythrocyte lysis and immunofluorescence staining. 950 87


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