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)

A combined diagnostic system for human papilloma virus (HPV) infections comprising the Papanicolaou test and in-situ hybridization assay was evaluated. Cervical smears from 259 women obtained with a "Cytobrush" were screened. Human papilloma virus genotypes 6/11, 16/18, 31/35/51 were detected by biotin in-situ hybridization in conjunction with a streptavidin-alkaline phosphatase detection complex. The diagnostic sensitivity of this assay was tested by human papilloma virus-DNA-positive human cervical carcinoma cell lines. According to the cytological (Bethesda system) and colposcopical criteria a random control group (n = 80) and prevention (n = 179) were chosen. Compared with Papanicolaou tests the frequency of human papilloma virus-DNA-positive cervices rose with the severity of cell abnormalities. The detection rate of human papilloma viruses-16/18 and human papilloma viruses-31/35/51 and of concomitant infections with human papilloma viruses-6/11 and human papilloma viruses-16/18 and/or human papilloma viruses-31/35/51 increased with the severity of cell dysplasia, whereas the rate of human papilloma virus-6/11 DNAs decreased. The incidence of oncogenic human papilloma virus types 16/18 and 31/35/51 rose with the age of the patients. A follow-up study by Papanicolaou tests of patients with mild (slight) and moderate dysplasias six months after human papilloma virus-DNA-hybridization indicates that human papilloma virus-16/18 DNA-positive lesions are more likely to persist or to progress than human papilloma virus-6/11 DNA-positive cell changes. Human papilloma virus-31/35/51 DNA-positive cell smears exhibited persistent behaviour. Our findings demonstrate that the Papanicolaou test combined with in-situ hybridization is suitable for early diagnosis and prevention of intraepithelial neoplasias and carcinomas of the uterine cervix.
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PMID:Papanicolaou test and enzyme-linked in-situ hybridization. A combined diagnostic system for papilloma virus infections with high prognostic value. 164 54

Combined application of a non-radioactive in situ DNA hybridization procedure and the immunoperoxidase technique on one tissue section is described. Of six potential protocols, only one proved to be successful. First, the immunohistochemical procedure including visualization of enzyme activity is performed; the in situ DNA hybridization protocol is then applied. Using this protocol, several antigens, detected with monoclonal antibodies, and target DNAs, detected by using biotinylated human cytomegalovirus or human papilloma virus type 16 DNA probes, could be distinguished by their peroxidase activity (brown precipitate) and alkaline phosphatase activity (purple-blue precipitate) respectively. The method allows immunophenotyping of virus-infected cells as well as simultaneous visualization of two viral parameters. This technique has important implications for research and diagnostic purposes.
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PMID:Simultaneous application of in situ DNA hybridization and immunohistochemistry on one tissue section. 247 18

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.
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PMID:Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes. 254 89

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.
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PMID:Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells. 254 32

A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.
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PMID:Interphase cytogenetics using biotin and digoxigenin labelled probes II: Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. 254 33

Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures.
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PMID:Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining. 255 31

Recently, sensitive non-isotopic in situ hybridisation (NISH) methodology for the detection of human DNA and human papilloma virus (HPV) DNA in archival paraffin blocks of cervix was described. An amended protocol, now used in this laboratory for detection of these genes by NISH is presented. The amendments include the following: protease digestion at 37 degrees C; tissue dehydration in air rather than ethanol; stringency washing in formamide solution; blocking non-specific binding of avidin alkaline phosphatase with a modified buffer; and increasing the concentration of avidin alkaline phosphatase for detecting low abundance DNA. These changes simplify and increase the sensitivity of the protocol such that "Y" chromosome repeats are visualised in almost all female cells.
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PMID:Non-isotopic detection of in situ nucleic acid in cervix: an updated protocol. 284 61

Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration of in situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100-200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method. The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.
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PMID:In situ hybridization: alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections. 332 63

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.
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PMID:HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. 812 21

A new DNA hybridization technique, based on chromatographic migration of DNA on a nitrocellulose strip passing through an immobilized probe area, is described. The new paper chromatography hybridization assay (PACHA) is faster and simpler to use than the conventional dot hybridization assay. In this assay, an aliquot of biotinylated, PCR-amplified target DNA is applied to one end of a nitrocellulose strip. The DNA migrates to the opposite end of the strip by capillary forces and hybridizes to a specific DNA probe immobilized in a reaction zone (RZ), located in the middle of the strip. Unhybridized DNA migrates away from the RZ. The biotinylated hybrid is visualized by a color reaction employing a streptavidin-alkaline phosphatase (SA-AP) conjugate and a specific chromogenic substrate. The new PACHA technique allows for detection of as little as 1-5 pg of specific human papilloma virus 16 (HPV16) DNA in 25 min of hybridization. In this system, the hybridization efficiency is controlled by the flow velocity of the hybridization solution (HS) and by the volume of the amplified labeled DNA migrating across the immobilized probe. Glycerol (30%) or polyvinyl pyrrolidone (PVP) (1%) reduces the flow rate by a factor of 2.5-3 and increases the sensitivity of the assay by a factor of 5.2 for glycerol and 2.6 for PVP. This novel method ensures efficient hybridization to multiple probes and appears to be superior to currently available solid-phase hybridization techniques.
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PMID:A novel rapid hybridization technique: paper chromatography hybridization assay (PACHA). 829 6


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