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At least 163 records · Page 9Linked to original sources
PubMed1995

Use of digoxigenin-labeled RNA probe to test hepatitis A virus antiviral drugs.

A nucleic acid hybridization assay was used to evaluate inhibitory activity of antiviral compounds against hepatitis A virus (HAV) in cell culture and compared to radioimmunoassay by analysis of variance procedure. The 5' genomic end of the HM-175 strain was used as digoxigenin-labeled RNA probe. Dot-blot examination showed a reduction of detectable HAV RNA in infected cells when treated with amphotericin B. An antiviral dose-effect was shown by statistical analysis of densitometric measures of hybridization signals. Comparison between molecular hybridization assay and radioimmunoassay by analysis of variance procedure showed the equivalence of both methods. Data previously obtained on selected drugs by antigen and infectious titres determinations were confirmed by hybridization assay and make possible digoxigenin-labeled RNA probe use to measure an antiviral dose-effect for screening of hepatitis A antiviral compounds.

Amides↗
PubMed1995

In situ localization with digoxigenin-labelled probes of tau-related mRNAs in the rat pancreas.

Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for the in situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR. Using these probes, an in situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells. The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas.

Animals↗
PubMed1995

The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids--an overview.

The use of non-radioactive nucleic acid probes has increased dramatically over the last years. The convenience of not having to deal with radioactive isotopes combined with the stability and economy of the non-radioactive systems has led to applications in various techniques. One of the most successful labelling and detection systems is based on the hapten digoxigenin. Here, the different methods for labelling nucleic acids with digoxigenin as well as the various possibilities for detection are described. Some typical applications illustrate the utility of the DIG system.

Animals↗
PubMed1995

Rapid in situ hybridization using digoxigenin probe and microwave oven.

In situ hybridization has been developed with probes labelled with a non-radioactive nucleotide, especially digoxigenin-coupled nucleotides. These non-radioactive probes significantly reduce safety problems and experimentation time. In this paper, we have studied by in situ hybridization the messenger RNA (mRNA) of the neuropeptide pro-opiomelanocortin (POMC), in the rat pituitary gland using digoxigenin labelled oligonucleotide and a microwave oven. Our protocol permitted us to visualize POMC mRNA in all cells of the intermediate lobe and a few corticotroph cells in the anterior lobe, as it has been already demonstrated and to complete the experiment in less than 24 hrs.

Animals↗
PubMedDate not supplied

[A comparative study of the action of digoxigenin-mono, bis- and tridigitoxosides in the cat heart-lung preparation (author's transl)].

In experiments using spontaneously beating and electrically driven cat hearts (heart-lung preparations), the inotropic effects and therapeutic ranges of digoxin and its bis- and monodigitoxosides were compared. The myocardial contractility was measured by the maximal rate of left ventricular pressure rise, with regard to heart rate, enddiastolic left ventricular pressure, and diastolic aortic pressure. The therapeutic range was defined as the ratio of the arrhythmogenic concentration to the threshold concentration. On molar basis, an increasing cardioactivity in the order digoxin, digoxigenin-bisdigitoxoside, digoxigenin-monodigitoxoside was found. The monoditoxoside displayed the largest therapeutic range. The therapeutic range of bis-digitoxoside was similar to that of digoxin. The findings suggest that cardiac glycosides, their cardioactive derivatives and their cardioactive metabolites, respectively, do not possess identical therapeutic ranges.

Animals↗
PubMed1995

Production of digoxigenin-labelled DNA probe for detection of Muscovy duck parvovirus.

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.

Animals↗
PubMed1993

Whole-Cell Hybridization of Frankia Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes.

Whole-cell hybridization with non-radioactively labeled oligonucleotide probes was used to detect and identify Frankia strains in pure cultures and in nodules. Digoxigenin-labeled probes, which were detected with antibody-alkaline phosphatase conjugates, were more suitable for in situ detection of Frankia strains than fluorescent probes since the sensitivity of the former was higher and problems arising from the autofluorescence of cells and plant material were avoided. Successful detection of Frankia strains in paraformaldehyde-fixed cell material with digoxigenin-labeled oligonucleotide probes depended on pretreatments to permeabilize the cells. Specific hybridization signals on vesicles were obtained after lysozyme pretreatment (1 mg ml for 30 min at 20 degrees C). Reliable penetration of the antibody-enzyme conjugate into hyphae required additional washing with the detergent Nonidet P-40 (0.1%) and toluene (1% in ethanol) after lysozyme treatment. Identification of Frankia vesicles in nodule homogenates was possible only after the removal of the polysaccharide capsule surrounding the vesicles. Incubation with H(2)O(2) (15% in water for 1 h at room temperature) before lysozyme and detergent treatments was found to facilitate specific hybridization. No filaments or spores could be detected in nodule homogenates. This technique should be a powerful tool in the identification of Frankia isolates, in the characterization of as-yet-uncultured nodule populations, and in the confirmation of the origin of unusual Frankia isolates.

Journal Article↗
PubMed1993

Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay.

An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.

Base Sequence↗
PubMed1991

In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs.

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.

Animals↗
PubMed1996

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes.

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

Animals↗
PubMed1998

The extent of proliferative and apoptotic activity in intraductal and invasive ductal breast carcinomas detected by Ki-67 labeling and terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling.

BACKGROUND: The balance among cell proliferation, cell differentiation, and cell death determines the cell number in a population as well as the size or even the stage of a tumor. Thus, to improve our understanding of the pathogenesis of neoplasms, it is important to investigate the regulation of both cell proliferation and cell death. METHODS: This study examined the occurrence of apoptosis and proliferative capacity in 46 breast carcinomas: 20 intraductal carcinomas (ductal carcinomas in situ [DCIS]) and 26 infiltrative ductal carcinomas (IDC). Terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) and immunostaining with the Ki-67 antibody were used in the examination. A ladder of DNA fragments induced by apoptosis was demonstrated by means of DNA agarose gel electrophoresis in 10 of the available TUNEL positive and negative samples. RESULTS: The results were correlated with p53, bcl-2, estrogen receptor (ER), and progesterone receptor (PR) protein expression, which would suggest association with apoptosis by immunohistochemistry. The apoptosis and proliferation of each cancer were expressed as the number of tumor cells undergoing apoptosis and proliferation per 1000 tumor cells. The extent of apoptosis was more frequently observed in DCIS than in IDC (21.9+/-6.8 vs. 4.0+/-0.9, P < 0.001), and the proliferation activity was significantly higher in IDC than in DCIS (16.8+/-6.5 vs. 3.5+/-0.8, P < 0.006). Apoptosis associated with MIB-1 positive cells and TUNEL labeling was significantly higher in IDC than in DCIS (3.26 vs. 0.42, P=0.001). In DCIS, apoptosis was correlated with p53 (r=0.663, P=0.005), and p53 had a reverse correlation with bcl-2 (r=0.620, P= 0.018). Moreover, bcl-2 expression was associated with ER (P=0.028) and PR (P= 0.005) expression in both DCIS and IDC. CONCLUSIONS: The results of this study show that a higher degree of apoptosis and lower proliferation activity in intraductal carcinoma result in a steady-state, self-renewing condition in which net growth of the tumor is rare. The results also indicate that apoptosis was altered by the expression of p53, bcl-2, ER, and PR.

Apoptosis↗
PubMed1996

Characterization of digoxigenin derivatives of human coagulation factors VIIa and X by electrospray mass spectrometry and enzyme kinetics.

Digoxigenin ester (Dig) derivatives of coagulation factors VIIa and X facilitate staining studies to localize tissue factor activity in human atherosclerotic plaques. The larger the number of attached Dig units the easier it is to form highly visual stains. Electrospray ionization was used to characterize the Dig derivatives, as a function of excess derivatization reagent, to determine the optimal derivatives, i.e. the highest number of Dig units attached with the product still retaining enzymatic activity. The enzymatic activity of factor VIIa derivatized at 50-fold Dig excess (with 28, 34, 48 or 52 Dig units attached, masses to 79 kDa) remained the same as that of native factor VIIa. In contrast, the enzymatic activity of factor X derivatives diminished above 15-fold Dig excess (15 Dig units attached, mass 65.6 kDa). The distribution of derivatized lysine, histidine, arginine, tryptophan and tyrosine residues was estimated for several possible configurations.

Amino Acids↗
PubMed1991

Quantitative non-radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin-labeled cRNA probes.

Non-radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non-radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol-induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin-11-UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin-alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 +/- 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low-resolution radioactive methods, showing a 30-80% increase in striatal preproenkephalin mRNA hybridization signal.

Animals↗
PubMed1995

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled, porcine male-specific DNA probe produced by polymerase chain reaction.

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5'-AAGTGGTCAGCGTGTCCATA-3' and 5'-TTTCTCCTGTATCCTCCTGC-3') for 236 bp fragment of porcine male-specific DNA sequence and 1.25 x 10(4) template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization.

Animals↗
PubMed1993

RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction.

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.

Acetylation↗
PubMed1994

Chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labeled oligonucleotides.

To establish a simplified, nonradioactive approach for identifying mRNAs on Northern blots, antisense oligonucleotides have been used as probes in combination with chemiluminescence-based detection. Oligonucleotides (approximately 32-mer) were end-labeled with digoxigenin (DIG) and used in conjunction with adamantyl 1,2-dioxetane aryl phosphate substrates (Lumigen PPD and CSPD). Oligonucleotides were designed as probes for several mRNAs in tissues of rats and mice, including the mitochondrial uncoupling protein, lipoprotein lipase, GLUT1, GLUT4, and beta-actin. Uncoupling protein mRNA was detected in total RNA from brown adipose tissue with a 32-mer DIG-labeled oligonucleotide, within 2 min of exposure to film. This mRNA could also be detected when as little as 250 ng of total RNA was applied to the gel, following 4 h exposure to film, and was present only in brown fat. The mRNA for lipoprotein lipase was detectable with a 30-mer DIG-labeled oligonucleotide in 1 micrograms of total RNA from mouse heart, within 2 h of exposure. The mRNA for the GLUT1 glucose transporter was detected in total RNA from rat midbrain using a 32-mer DIG-labeled oligonucleotide, while beta-actin mRNA was detected with a 30-mer oligonucleotide. The mRNA for the insulin-sensitive glucose transporter GLUT4 was detected with a 32-mer DIG-labeled oligonucleotide and found only in those tissues in which glucose uptake is stimulated by insulin. The speed of detection was greater with CSPD and was augmented by exposure of membranes to film at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

Quantitation of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots.

Quantitation of message from low-abundance mRNAs and limited availability of tissues requires sensitive methods for probe detection, accurate methods for quantitation of signal, and the ability to strip and reprobe membranes. A random-primed, digoxigenin-labeled probe from cDNA of FMO1, an isoform of the flavin-containing monooxygenase gene family, from rabbit was used in the evaluation and optimization of the Genius system for quantitation of signal from DNA and RNA slot blots. Criteria for optimization were a low signal to noise ratio, a linear increase in density of signal vs nuclei acid concentration of bands on X-ray film, complete stripping of membranes, and reproduction of the initial banding pattern upon rehybridization. A low signal-to-noise ratio was obtained with an aqueous prehybridization/hybridization solution. DNA slot blots were successfully quantitated before and after alkaline stripping from positively charged membranes. RNA slot blots were subject to excessive and uneven loss of RNA from the membranes during stripping procedures. Reliable quantitation for more than one cycle of detection required highly charged nylon membranes, and careful tailoring of RNA fixation methods and alkaline stripping conditions.

Animals↗
PubMed1994

Analysis of microsatellite DNA using a non-radioactive method: blotting from polyacrylamide gel to nylon membrane using a gel dryer with subsequent hybridization to digoxigenin-labelled (dCdA)8 oligonucleotide probes.

A non-radioactive method for the analysis of microsatellite DNA polymorphisms (mainly (CA)n-repeats) is described. Variation in length of (CA)n and (GT)n blocks was demonstrated directly by amplification of DNA from within and immediately flanking the repeat blocks using the polymerase chain reaction. The amplified DNA was resolved on a denaturing polyacrylamide gel, transferred to a nylon membrane using a gel dryer, and subsequently hybridized with digoxigenin-labelled (dCdA)8 oligonucleotide probes. This method enables easy and rapid analysis of polymorphic microsatellite DNA.

Acrylic Resins↗