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)

Plasmids were constructed whereby the expression of a reporter gene, either the cDNA corresponding to the secreted form of human alkaline phosphatase (SEAP) or the herpes simplex virus type 1 (HSV1) thymidine kinase (tk) gene, was rendered dependent upon the expression of the human immunodeficiency virus type 1 (HIV1) tat and rev proteins. The SEAP or tk genes were placed between HIV1 splice donor and acceptor sites. One SEAP construct carried a series of alternating splice donor and acceptor sites. In all cases, the rev response element mapped within an intron. Despite such mimicry of the HIV1 genome, residual expression of the reporter gene in the absence of tat and rev was observed. These results, as well as non-specific T-cell recruitment, suggest limits to the specificity of using HIV-activated toxic gene expression to kill HIV-infected cells.
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PMID:Residual expression of reporter genes in constructs mimicking HIV genome organization. 748 Oct 89

In this paper, the synthesis of new phosphonoacetic acid derivatives and their applications in fields of biotechnological interest are discussed. Phosphonoacetic acids are competitive inhibitors of alkaline phosphatase, an enzyme widely used in diagnostics, as colorimetric detection tool. The phosphonoacetic acid's inhibition activity has been exploited by us for the obtainment of an innovative technique for non-radioactive DNA probes detection, the last being based on DNA labeling with the enzyme inhibitor, followed by detection by means of the chromogenic enzyme and substrate. Moreover, we have found a further application of phosphonoacetic acids, by the preparation of an affinity chromatography support that has been revealed to be very effective in the purification of alkaline phosphatase. Finally, phosphonoacetic acid derivatives have been tested also for their antiviral activity. Some of them, examined in preliminary in vitro experiments, have been found very active against Herpes simplex virus.
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PMID:Synthesis and applications of phosphonoacetic derivatives. 776 51

The mode of formation and role of rheumatoid factors (RF) in transplantation were reviewed. RF might be produced through stimulation with altered transplantation antibody, which undergoes conformational changes resulting from the reaction with a graft. Some recipients might have such sources of infection as cytomegalovirus or human herpes virus 6 (HHV-6), because almost all patients were treated with immunosuppressive agents. Many bacteria and viruses have IgG Fc receptors on their surface. Herpes virus infection, such as herpes simplex virus, has been demonstrated to be capable of expressing Fc receptor on the infected cells. These Fc receptors could produce RF through the response of antibody production to anti-idiotype of Fc receptors. Also, IgG, which binds to Fc receptors, changes conformationally to produce RF. RF can also be produced as a result of graft versus host reaction. Although IgG RF reacting with homologous IgG is believed to have the most important role in pathogenic conditions, they have not been studied because of difficulties in their measurements. We developed an enzyme immunoassay for detection of IgG RF combining with homologous IgG. The wells of an assay plate were coated with Fc fragment of human IgG, and after incubation with tested serum, binding of IgG RF to the Fc was detected by means of goat anti-human IgG F (ab')2 antibodies followed by alkaline phosphatase conjugated rabbit anti-goat IgG and substrate. IgG RF were increased significantly more in transplantation sera of patients who rejected grafts than in those of patients who had satisfactory graft function. Some evidence for the role IgG RF in rejection, even if only circumstantial, has been provided.
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PMID:Rheumatoid factor: a review of the mode of formation and role in transplantation. 812 26

The herpes simplex virus type 2 (HSV-2, strain 333) UL3 open reading frame (ORF) codes for a protein with a predicted molecular weight of 29,681. Comparisons of the UL3, strain 333 ORF show essentially complete amino acid identity with HSV-2 (strain HG52) and a 75% amino acid identity with HSV type 1 (strain 17). To characterize the expression of this gene, a hydrophilic region of the HSV-2 UL3 gene was cloned into a bacterial expression vector. The resulting fusion protein was used to generate antibodies in rabbits. In vitro translation of HSV-2-derived mRNA followed by immunoprecipitation with the rabbit antisera reveals a major 28,000-Da protein as judged by SDS-polyacrylamide gel electrophoresis. This is consistent with the predicted molecular weight of an unmodified UL3 protein. Pulse-labeling of infected cells, with [35S]methionine, followed by immunoprecipitation and electrophoretic analysis reveals three distinct bands of 28,000, 30,500, and 33,000 Da. Labeling infected cells with [32P]orthophosphate shows that the 30,500- and the 33,000-Da species are post-translationally phosphorylated. The 30,500-Da species can be converted to the 28,000-Da species with alkaline phosphatase treatment. Interestingly, the 33,000-Da species is resistant to this treatment. Immunohistochemical analysis of infected cells reveals that the UL3 protein has a perinuclear location early in infection and at later times becomes associated with the nucleus as discrete particles. A mutant of HSV, which has a major deletion of the UL3 coding region does not show any immunohistochemical staining with the UL3 antisera. The wild-type virus-infected cell staining pattern remains the same subsequent to DNAse and RNAse treatment, indicating that the UL3 protein product is not directly associated with nucleic acid.
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PMID:The herpes simplex virus type 2 UL3 open reading frame encodes a nuclear localizing phosphoprotein. 833 18

Detection of herpes simplex virus (HSV) by a newly developed HSV-DNA in situ hybridization (ISH) technique (Diagnostic Hybrids, Inc., Athens, OH, USA) was compared with a reference standard that combines observation for cytopathic effect in shell vial cultures with subsequent identification of virus by staining with fluorescein-labeled HSV-specific monoclonal antibody. The new technique utilizes a probe consisting of an alkaline phosphatase direct labeled, cloned, single stranded DNA fragment that is common to HSV-1 and HSV-2. Both methods include enhancement of infection by centrifugation. In concurrent testing of 98 freshly collected specimens, HSV was detected in 17 by culture and 16 by ISH. In testing of 57 frozen positive specimens, HSV was detected in 54 by culture and 53 by ISH. Of the total isolates, 22 were HSV-1 and 49 were HSV-2. HSV was detected after 24 hrs of incubation by the DNA probe technique. Using the shell vial method as a reference standard, the new HSV-DNA ISH method had a sensitivity of 97.2%, specificity of 100%, positive predictive value of 100%, and a negative predictive value of 97.7%. HSV-DNA ISH appears to be a practical, sensitive, and specific technique for detection of HSV.
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PMID:Rapid detection of herpes simplex virus in culture by in situ hybridization. 838 92

Eighteen patients, ten affected by pemphigus vulgaris and four affected by herpes simplex of the oral mucosa, together with four healthy patients as controls, were investigated by cytologic examination of Papanicolaou stained smears obtained by scraping the oral mucosa. In all cases additional smears were immunostained with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique using monoclonal antibodies against human heavy IgG chains and lambda light chains. The results have shown that, for a cytological diagnosis of pemphigus, this technique can be used as an easy substitute for the immunofluorescence test and does not require any specialized training or equipment. The findings are clearly detectable by light microscopy and allow, together with the immunostaining, an adequate visualization of cell morphology.
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PMID:Immunocytochemical detection of autoantibody deposits in Tzanck smears from patients with oral pemphigus. 923 84

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

We generated neurotropic herpes simplex type 1 viruses expressing human placental alkaline phosphatase and studied the utility of this enzyme as a marker of infected neurons. The neurotropism of these viruses was assessed by their ability to infect sympathetic preganglionic neurons after adrenal injection in hamsters. The transneuronal transfer of these viruses was examined by their ability to cross the peripheral synapse from the kidney to renal preganglionic neurons or to cross the central synapse from the adrenal gland to the medulla oblongata. Finally, we injected an alkaline phosphatase-expressing herpes simplex virus into the adrenal gland and a beta-galactosidase-expressing herpes simplex virus (US5gal) into the muscular wall of the small intestine to label two neural circuits in one animal and to assess the feasibility of a dual-virus labelling system. The alkaline phosphatase gene was inserted into the glycoprotein J locus or the virus-induced host shut-off locus in the herpes simplex genome to create viruses which replicate (gJHAP HSV or vhsHAP HSV) or into the thymidine kinase locus to generate a virus that does not replicate in neurons in vivo (TK- HAP HSV). Each of the three viruses was retrogradely transported from the adrenal gland of hamsters to sympathetic preganglionic neurons, suggesting that the neurotropism of these viruses was maintained. gJHAP HSV travelled transneuronally from the kidney to sympathorenal preganglionic neurons and from the adrenal gland to neurons in the rostral ventrolateral medulla. Neuronal infection with alkaline phosphatase-expressing virus could be identified using histochemistry but detailed morphology of these neurons was not revealed. However, staining by anti-herpes simplex virus immunoperoxidase demonstrated that they had normal morphology. Identification of two distinct neural circuits in one animal was achieved with our dual-virus labelling system. The nonreplicating TK- HAP HSV was used in combination with US5gal to identify intestinal and adrenal sympathetic preganglionic neurons. The beta-galactosidase-expressing intestinal neurons were labelled bilaterally in the nucleus intermediolateralis, pars principalis, and alkaline phosphatase-expressing adrenal neurons were found ipsilaterally. Some clusters of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis contained mostly intestinal sympathetic preganglionic neurons and a few adrenal sympathetic preganglionic neurons. In other areas, the opposite pattern occurred. About 3-7% of the labelled sympathetic preganglionic neurons were double-labelled by both markers. The distinct and crisp morphology and dendritic processes of neurons stained by beta-galactosidase histochemistry contrasted with the partial staining of neurons by alkaline phosphatase, revealing beta-galactosidase as a better marker of infected neurons. In conclusion, alkaline phosphatase-expressing herpes simplex viruses are yet neurotropic after insertion of this marker enzyme into any of three different loci of the herpes simplex genome. One replicating alkaline phosphatase-expressing virus travelled transneuronally. These alkaline phosphatase-expressing herpes simplex virus can be used together with beta-galactosidase-expressing herpes simplex viruses to determine the target specificity of sympathetic preganglionic neurons controlling visceral organs or can be used to express two different recombinant genes in two targeted neuronal populations. This study suggests that sympathetic preganglionic neurons controlling the intestine and adrenal gland are almost completely distinct.
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PMID:Simultaneous identification of two populations of sympathetic preganglionic neurons using recombinant herpes simplex virus type 1 expressing different reporter genes. 946 44

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [35S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.
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PMID:Varicella-zoster virus (VZV) ORF32 encodes a phosphoprotein that is posttranslationally modified by the VZV ORF47 protein kinase. 973 48

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.
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PMID:In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity. 1119 54


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