Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytoplasmic inclusion bodies corresponding in their tinctorial properties to Negri bodies were detected in TEp-2/2 and BHK-21/13S cell cultures chronically infected with fixed rabies virus. The number of cells containing the inclusions was always than that of the cells producing virus-specific antigen. Histological examinations of chronically infected cultures revealed considerable inhibition of the acid phosphatase activity, some weakening of the reaction to alkaline phosphatase, and a marked decline in the activity of succinate dehydrogenase. The intensity of reaction to RNA in chronically infected cultures was increased, particularly in those zones of the cells where RNA-containing inclusions were detected. The activity of the respiratory enzyme HAD-H2 tetrasolium reductase in HEp-2/2 cells was reduced and in BHK-21/13S cells increased as compared to the control.
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PMID:[Cytologic and histochemical study of cell cultures chronically infected with fixed rabies virus]. 35 98

One of the major structural differences between rabies virus and vesicular stomatitis virus (VSV) is that the nucleoprotein (N) is the major phosphoprotein and the nominal phosphoprotein (P) is less phosphorylated in rabies virus, whereas P is the major phosphoprotein and N is not phosphorylated in VSV. We investigated the function of phosphorylation of rabies virus N after dephosphorylation of N with alkaline phosphatase or after changing the phosphorylated serine at position 389 to alanine by site-directed mutagenesis. The unphosphorylated N, in comparison to the phosphorylated N, was studied for its abilities to encapsidate rabies virus leader RNA and to support transcription and replication of a rabies virus minigenome. We found that unphosphorylated N binds more strongly to leader RNA than the phosphorylated N; however, the rates of transcription and replication of the rabies virus minigenome were significantly lower with the unphosphorylated N than with the phosphorylated N. This indicates that the phosphorylation of rabies virus N plays an important role in the regulation of rabies virus transcription and replication, probably via modulation of leader RNA encapsidation.
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PMID:Phosphorylation of rabies virus nucleoprotein regulates viral RNA transcription and replication by modulating leader RNA encapsidation. 988 76

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).
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PMID:Studies on the rabies virus RNA polymerase: 2. Possible relationships between the two forms of the non-catalytic subunit (P protein). 988 49

The purpose of the present study was to implement a fluorometric method for detecting and quantifying viral antigens in human meduloblastoma cells infected by two types of fixed rabies virus (CVS-MB and CVS-BHK) and a street virus using a cell-enzyme linked immunosorbent assay (cell-ELISA) technique; alkaline phosphatase was used as the antibody-marker enzyme and 4-methyl-umbelliferyl-phosphate as the fluorogenic substrate. The system was used for detecting up to 1:10,000 viral inoculums, followed by evaluating the effect of heparin on infection. Infected cultures were reliably differentiated from their respective negative controls in both assays allowing data to be analysed statistically. As reported in another study, heparin produces strong inhibition when the CVS-BHK viral strain is used for infection; it has thus been suggested that it binds to the neural cell adhesion molecule and could be blocked by using this drug. This fluorometric method is less time-consuming, has increased reproducibility and useful for quantitation of collected data and can therefore be considered as a useful tool for research.
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PMID:Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition. 1589 63

We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NC-like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'- and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein.
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PMID:Structural difference recognized by a monoclonal antibody #404-11 between the rabies virus nucleocapsid (NC) produced in virus infected cells and the NC-like structures produced in the nucleoprotein (N) cDNA-transfected cells. 1611 4

The binding of rabies virus to cellular membranes was measured using an enzyme-linked immunosorbent assay (ELISA). Virus binding to membranes adsorbed to the wells of microtiter plates was detected with rabies virus antibody and alkaline phosphatase-linked second antibody. The greatest degree of binding was to myotube, neuroblastoma, and salivary gland membranes; intermediate levels occurred in striated muscle and nerve membranes; and low levels of binding were found in other membranes, including those of most parenchymal organs. Binding of rabies virus to myotube membranes was saturable, dependent on pH (with an optimum of pH 6.0), facilitated by the divalent cations Ca++, Mn++, and Mg++, and was temperature dependent. Binding was greatly reduced by inactivation of virus with beta-propiolactone or treatment of virus with trypsin. In embryonic chick myotubes, total acetylcholine receptor content and acetylcholinesterase activity undergo marked changes during development, first increasing and then decreasing at the time of hatching. Binding of rabies virus followed a similar pattern, indicating that the virus may interact with the acetylcholine receptor or other surface molecules undergoing similar developmental changes.
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PMID:Rabies virus binding to cellular membranes measured by enzyme immunoassay. 1675 1

The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.
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PMID:Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture. 1784 21

The most widely used test for rabies diagnostics is the fluorescent antibody test, which is recommended by both the World Health Organization and the World Organisation for Animal Health (OIE). This test may be used directly on a smear, and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue for diagnosis. The colorimetric enzymes are usually coupled to an antibody by chemical means using cross-linking reagents. However, such non-specific procedures lead to heterogeneous conjugates, sometimes with reduced activity and specificity. To bypass these problems, genetic engineering has provided a way to create chimeric bifunctional molecules in which the variable domains of an antibody are genetically linked to unrelated protein tracers. In this study, we describe the successful production of a bifunctional chimeric protein based on alkaline phosphatase-fused anti-rabies virus glycoprotein scFv antibody fragment. We also report the antigen binding properties and the alkaline phosphatase activity of the recombinant conjugate protein. We established its value as a novel in vitro tool for detecting the rabies virus in brain smear in a one-step procedure; it presents a similar sensitivity and specificity to that obtained using standard reagents.
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PMID:Genetically engineered colorimetric single-chain antibody fusion protein for rapid diagnosis of rabies virus. 1863 11