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 chemiluminescent dot-blot hybridization assay was developed for the detection of Muscovy duck parvovirus (DPV) by using a non-radioactive DPV DNA probe. A 1030bp HindIII-Bg/II fragment of DPV DNA was labelled with digoxigenin-labelled dUTP. The hybridized DPV DNA probes were detected by an immunoenzymatic reaction using anti-digoxigenin-antibody Fab fragments conjugated to alkaline phosphatase and visualized by chemiluminescent reaction. The assay proved to be sensitive since up to 3 fg of homologous DPV DNA and 10(0.4) EID50 mul-1 of DPV infected amino-allantoic fluid could be visualized. It appeared to be specific for the detection of different strains of DPV and Derszy's disease virus (DDV). Nevertheless, the dot-blot assay showed a lower sensitivity to detect DDV infected samples. No hybridization was noticed between DPV DNA probe and the two mammalian parvovirus strains tested (canine and porcine parvoviruses), emphasizing nucleotidic sequence heterologies. The use of the probe for DPV diagnosis purpose is discussed. To our knowledge, this work constitutes the first description of a dot-blot hybridization assay for the detection of an avian parvovirus.
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PMID:Production of digoxigenin-labelled DNA probe for detection of Muscovy duck parvovirus. 776 Aug 58

Recent evidence suggests that anti-androgen therapy may be useful in patients with androgen receptor (AR)-positive hepatocellular carcinomas (HCC), as determined by a steroid binding assay. To evaluate the AR expression of HCC, in both histological and cytological material, we developed a non-radioisotopic in situ hybridisation (NISH) assay specific for the human AR mRNA. A synthetic oligonucleotide complementary to positions 661-695 of the human AR coding sequence was end-labelled with digoxigenin-dUTP and revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. We analysed 22 formalin-fixed, paraffin-embedded HCC, obtained at surgery, together with the corresponding non-neoplastic liver tissues (19 cases). In six cases, cell blocks obtained by fine-needle aspiration (FNA) prior to surgery were also available. Positive controls included seminal vesicles and prostate tissues. Sixteen HCCs (73%) expressed a variable amount of AR mRNA, with the proportion of positive cells ranging from very few to more than 90%. Normal hepatocytes were stained weakly and focally in eight cases (42%). Appropriate controls, inclusive of immunohistochemical detection of the AR protein in selected cases, established the specificity of the assay. Data obtained on FNA specimens were predictive of the results on histologic material. However, in two cases the NISH assay was negative on the cytological specimen but stained rare hepatocytes within the surgically resected tumor. In conclusion, NISH is a novel procedure for rapid and specific assessment of the expression of AR in HCC tissue. Its clinical significance, in terms of predictivity of response to anti-androgen treatment, needs to be assessed in large correlative studies.
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PMID:Detection of human androgen receptor mRNA in hepatocellular carcinoma by in situ hybridisation. 796 81

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.
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PMID:Detection of bacteria in phagocyte-smears from septicemia-suspected blood by in situ hybridization using biotinylated probes. 796 83

We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
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PMID:Detection of HIV-1 DNA by polymerase chain reaction incorporation of digoxigenin-11-dUTP and hybridization to immobilized probes. 796 98

In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed, paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.
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PMID:PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. 798 96

A tendon is predominantly composed of collagen Type I. To determine the synthesis of collagen Type I after a rotator cuff tear, an in situ hybridization technique was applied to localize cells containing procollagen alpha 1 Type I in the proximal stump of five torn supraspinatus tendons obtained during surgery. Specimens were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 6 microns. A 22 mer oligonucleotide corresponding to a sequence coding a part of human procollagen alpha 1 Type I messenger RNA (mRNA) was used as a hybridization probe. The probe was 3'-end labeled with digoxigenin-11-dUTP, and the probe-mRNA hybrids were enzymatically visualized using conventional chromogens for alkaline phosphatase. The procollagen alpha 1 type I mRNA was clearly observed in the cells near the margin of the tear. However, they were not consistently found in the vicinity of the intratendinous extension of the tear and in cells of the subacromial bursa. It is concluded that this method should be used to study the characteristics of collagen synthesis in a torn rotator cuff tendon.
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PMID:Localization of mRNA of procollagen alpha 1 type I in torn supraspinatus tendons. In situ hybridization using digoxigenin labeled oligonucleotide probe. 802 Feb 12

In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.
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PMID:Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation. 822 21

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.
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PMID:Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase. 822 79

The distribution of uteroglobin mRNA has been investigated in the endometrial compartments of the rabbit uterus during early pregnancy (day 0.5 p.c.--day 12 p.c.) using nonradioactive in situ hybridization. Digoxigenin-dUTP labeled oligodesoxy-nucleotide-probes (24mer) and an anti-digoxigenin-antibody conjugated with alkaline phosphatase were developed and used. It could be shown, that uteroglobin mRNA localization is restricted to the endometrial epithelium. There are differences in the extent of uteroglobin mRNA expression within the epithelial cells, which is in accord with our interpretation on the existence of different epithelial cell populations. From day 0.5 p.c. to day 9 p.c. the cells of the basal glands express uteroglobin mRNA continuously, whereas the proliferating surface epithelium shows a remarkable fluctuating pattern of uteroglobin mRNA expression. On day 2 p.c. the whole surface epithelium starts to express the uteroglobin message, and up to day 5 p.c. all these cells show a high level of uteroglobin mRNA expression, which drops significantly on day 6 p.c., when significant changes in the cyto-morphology of the surface epithelium for implantation occur. On day 7 p.c., there is no more uteroglobin mRNA expression in the surface epithelium, however remaining expression in the basal glands. The latter is evident up to day 9 p.c. From day 10 p.c. onwards, neither the luminal nor the deep glandular epithelium express any uteroglobin mRNA. Our observations on the cellular level have been continued in parallel studies on endometrial homogenates by Northern Blot analysis of uteroglobin mRNA (600 bases). Finally, it is discussed whether Uteroglobin mRNA may be an useful marker for the differentiation of various endometrial epithelial cell populations.
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PMID:Localization of uteroglobin mRNA by nonradioactive in situ hybridization in the pregnant rabbit endometrium. 830 87

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.
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PMID:Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. 834 53


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