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Biomedical subjects

A K Raap

Publications and source records attributed to A K Raap.

At least 145 records · Page 8Linked to original sources
PubMed1989

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

2-Acetylaminofluorene↗
PubMed1988

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization.

The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material. A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as "ectopic" CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral ganglion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors.

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.

Carcinoma↗
PubMed1988

Non-radioactive in situ hybridization for the detection of cytomegalovirus infections.

Acetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection, detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.

2-Acetylaminofluorene↗
PubMed1988

Immunofluorescence detection of herpes simplex virus type 1 antigen in cerebrospinal fluid cells of experimentally infected mice.

Experimentally infected mice were used to assess the value of immunofluorescence (IF) procedures in cerebrospinal fluid (CSF) cells in comparison with routine viral culture of CSF for diagnosis of herpes simplex type-1 (HSV-1) encephalitis. Virus specific antigen was detected by immunofluorescence in the majority of CSF cells in 75% of infected animals. In contrast, only 20% of infected mice had positive viral cultures, which sometimes took as long as a week to show a cytopathological effect (CPE). It is concluded that antigen detection by immunofluorescence is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in CSF cells of experimentally infected mice. Our results put forward a plea for further studies using these techniques on CSF samples from patients with suspected HSV-1 encephalitis. The prerequisites for reliable interpretation of results have been defined in this study.

Animals↗
PubMed1988

Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent.

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Acetyltransferases↗
PubMed1987

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts.

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.

Animals↗
PubMed1986

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures.

With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes.

Acridine Orange↗
PubMed1986

Localization properties of fluorescence cytochemical enzyme procedures.

Fluorescence enzyme cytochemical procedures will contribute significantly to biomedical problems where knowledge of the enzymic composition of individual cells is important. Compared with the number of absorbance enzyme cytochemical methods, relatively few fluorescence procedures have been reported. In this paper, the merits of the described methods are discussed. A distinction is made between methods with and without a capture reaction. Only a few methods satisfy the requirement of accurate localization of the final product and high signal to noise ratios. Thus, there still is a need for valid fluorescence cytochemical enzyme methods. It is concluded that the bottle neck for valid fluorescence cytochemical enzyme methods is the development of efficient fluorogenic capture reactions for the primary enzyme products.

Animals↗
PubMed1986

Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immobilized on nitrocellulose.

The nitrocellulose binding assay was used for quantitative studies on the cytochemical reactions for the three enzymes most frequently used in immunocytochemistry. The results show a linear relationship between the amount of enzyme immobilized on nitrocellulose and the amount of the enzyme reaction product. The similar course of the formation of the reaction product after DAB/H2O2 staining for peroxidase immobilized on nitrocellulose and for immunoperoxidase labeled cells indicates a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. Furthermore, a mild acid treatment for the abolition of endogeneous peroxidase activity in tissues and cells applicable to immunoperoxidase staining procedures is proposed.

3,3'-Diaminobenzidine↗
PubMed1986

Bi-color detection of two target DNAs by non-radioactive in situ hybridization.

A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the procedure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of non-radioactive in situ hybridizations.

Animals↗
PubMed1986

Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition.

Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.

Animals↗
PubMed1985

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry.

Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3'-diaminobenzidine (DAB), p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co2+ and Ni2+ ions. The results indicate that DAB-imidazole and DAB-Co2+ and Ni2+ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.

Alkaline Phosphatase↗
PubMed1985

Hybridocytochemistry as a tool for the investigation of chromatin organization.

The study of nuclear components in cells and tissues has resulted in a wealth of information with regard to the role of chromatin in cellular processes. Here, a survey is given of procedures which allow the cytochemical investigation of nucleic acid present in microscopic preparations of cells, nuclei or metaphase chromosomes. Special attention is given to recent developments in hybridocytochemistry (in situ hybridization) which facilitate microscopic identification and localization of specific nucleotide sequences within the total amount of nucleic acids present. Some of the potentialities and limitations of these in situ hybridization methods are discussed.

Animals↗
PubMed1984

Anti-DNA.RNA sera. Specificity tests and application in quantitative in situ hybridization.

The specificity of anti-nucleic acid antisera can immunocytochemically be evaluated with test systems which apply various nucleic acids immobilized to inert matrices. When using polyrA.polyrU as a model compound for dsRNA, it is important to prevent the formation of the triple stranded form polyrA.(polyrU)2. Anti-dsRNA antibodies which, when tested with the correct test system, proved to be present in an earlier described anti-DNA.RNA serum, could be removed by adsorption. By cytofluorometric comparison of the immuno-fluorescence signals obtained with the anti-dsRNA containing serum and the absorbed serum, it could be shown that the anti-dsRNA antibodies do not contribute to the specific signals measured after in situ hybridization. Repetitive incubations with the anti-DNA.RNA serum led to a considerable increase in immunofluorescence signal.

Animals↗
PubMed1984

Localization mechanisms in enzyme cytochemistry studied with alkaline phosphatase-loaded erythrocyte ghosts.

Erythrocyte ghosts containing varying amounts of alkaline phosphatase were used to study the localization mechanisms of three metal salt and one azo method for this enzyme. For the azo method, the minimal amount of alkaline phosphatase that can be visualized within the ghosts proved only to be limited by the optical properties of the azo compound. In contrast, for the metal salt methods, a certain threshold activity had to be present in the ghosts in order to obtain correct localization of the final reaction product. The localization properties of both azo and metal salt methods conformed to the theories of cytochemical enzyme localization presented to date. By determining the rate constant of the capture reaction and the diffusion constant of the primary product, the localization properties of the azo method could be predicted. Some remaining discrepancies between theory and practice are discussed.

Alkaline Phosphatase↗