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P van Duijn

Publications and source records attributed to P van Duijn.

At least 19 recordsLinked to original sources
PubMed1989

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells.

The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.

Animals↗
PubMed1986

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of the mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten.

The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.

Animals↗
PubMed1986

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. II. Effects of variations in ligand structure on the in situ detection of mercurated probes.

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achieved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand. In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands. The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.

Animals↗
PubMed1979

Microscopic cytochemistry as matrix chemistry.

Microscopic cytochemical procedures, unlike reactions in test tubes, have to be undertaken while the compounds to be localized and quantified are present in the structural matrix of cells or tissues. Cytochemical reactions therefore differ from analogous staining reactions in homogeneous media because the diffusion of reagents into and out of the matrix and the molecular state around the compounds in the matrix to be stained, as well as the presence of many potentially interfering substances in the matrix, produce additional complications in terms of both specificity and quantitation. These complications can be studied quantitatively and in detail in artificially prepared matrices of defined geometry into which pure compounds or mixtures (of known composition) with other biological compounds can be incorporated. Since the composition of the matrix models is known, and the amount or activity of the compound to be stained can be analysed biochemically too, matrices are well suited to studies of the specificity of cytochemical reactions. They can also be used to study quantitative aspects of the influence of fixation procedures on the state of the incorporated compound. In addition, matrices can--by further biochemical analysis--be used to calibrate the intensity of cytophotometrically measured staining in terms of amounts of stained substance or in biochemical enzymic activity units. Finally, films and beads containing known amounts of known compounds can be used for objective quality control of commercially available cytochemical reagents.

Alkaline Phosphatase↗
PubMed1979

Reflection versus fluorescence: a note on the physical backgrounds of two types of light microscopy.

Bright microscopic images against a dark background can be originating not only from fluorescence, but also from selective reflection. Selective reflection or scattering of visible light in microscopic preparations can be used for the visualization of sometimes otherwise barely distinguishable material. The images obtained superficially resemble those from fluorescence microscopy. They do not, however, result from huminescence but from selectively reflected light with wavelengths in the region of the absorbance peak of the chromophore present in the stained biological material. The respective backgrounds of the underlying physical phenomena and the conditions under which selective reflection can occur are discussed.

Light↗
PubMed1978

Enzyme layers on glass as a new model for the quantitative study of capture reactions in cytochemistry, with special attention to acid phosphatase.

A model system is described for the study of capture reactions for diffusable compounds in enzyme cytochemistry. The model, which allows the investigation of the influence of the composition of the cytochemical medium, the enzymatic activity, and the dimensions of the enzymatic site on the capture reaction, consists of very thin homogeneous layers of enzyme (0.01-0.1 micrometer thick) on glass, which are incubated in the cytochemical medium. The fraction of the total amount of liberated product precipitated in the enzyme layer is dependent not only on the trapping efficiency of the cytochemical medium but also on the concentration of the primary reaction product that can be built up in the enzyme layer. Calculations were performed to determine the steady-state concentration of the primary reaction product that can be built up in the enzyme layer. Acid phosphatase was used as enzyme. The problems associated with the model and its applicability to other types of cytochemical reactions are discussed.

Acid Phosphatase↗
PubMed1978

Effect of ozone on lysosomal enzymes of alveolar macrophages engaged in phagocytosis and killing of inhaled Staphylococcus aureus.

The role of lysosomal enzymes in the inactivation of inhaled bacteria by alveolar macrophages was studied in rats infected with aerosols of Staphylococcus aureus and then exposed for 5 hr to 2.5 ppm of ozone to determine whether pollutant-induced defects in phagocytic killing were associated with reduction in enzyme activity. Rates of bacterial ingestion and the activities of cellular acid phosphatase and beta-glucuronidase were measured simultaneously in in situ perfused right lungs by sequential staining of frozen sections for enzyme and bacteria. Quantitative measurements of enzyme activity within macrophages without ingested bacteria were made with a computer-controlled cytospectrophotometry system. Exposure to ozone resulted in diminished rates of bacterial clearance and ingestion, large increases in numbers of intra- and extracellular staphylococcal microcolonies, and an absence of enzyme activity for macrophages containing bacterial microcolonies. Enzyme activity was unimpaired in macrophages without ingested bacteria. These results, in which absence of enzyme activity occurred only in macrophages subjected to the dual insults of ozone exposure and ingested bacteria, prove a relationship between impairment in bactericidal capacity and cellular activities of lysosomal enzymes.

Acid Phosphatase↗
PubMed1978

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. I. Description of the model with special attention to phosphate capture in acid phosphatase cytochemistry.

A model system is described for the investigation of the dynamics of precipitation processes in a matrix. In this system a solution containing the molecular species to be precipitated and the precipitating medium are pumped along opposite sides of a polyacrylamide film. The solutions flowing continuously along the film, interact and can form a precipitate inside the film. The applicability of the model was tested on the capture reaction for phosphate ions by the Gomori type medium for acid phosphatase. Precipitation of lead phosphate in the film occurred only at a phosphate concentration above a certain value. The dependence of this minimal phosphate concentration on various parameters was studied and the results were compared with values found in earlier model studies and calculations concerning phosphate concentrations that can be built up in lysosomes during the Gomori reaction. The system seems promising for obtaining fundamental data about other cytochemical enzyme trapping reactions as well as for the matrix facotrs involved in bone calcification and shell formation.

Acid Phosphatase↗
PubMed1977

A new type of cytocentrifuge, the valve-centrifuge.

A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves. The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation. Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei thorugh gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.

Animals↗
PubMed1977

High-resolution scanning-densitometry of photographic negatives of human chromosomes. III. Determination of fluorescence emission intensities.

Principles and techniques are discussed for measuring with high topological resolution local emission in fluorescing objects, using photographic negatives. Determination of fluorescence intensities is only possible when an unequivocal relation between the original local fluorescence emission intensities of the object, and the transmittances or densities recorded in the microfluorophotograph is known. This relation is formulated in the theoretical part. From this relation it can be concluded that the recorded intensities can be measured optimally when the optical density values produced by the fluorescence emission fall in the range of the linear portion of the Hurter and Driffield curve. In order to obtain this situation, a uniform low-level pre-exposure of the film emulsion to (white) light is carried out prior to the actual fluorescence emission exposure. This pre-exposure acts to elevate the signal exposure to the linear (steeper) part of the H.D. curve. Inhomogeneity of the excitation beam in the object field, or differences in film emulsion response to the light exposure, will result in erroneous optical densities recorded in the photographic negative. Correction for such artifacts could be obtained by addition of a low concentration of fluorophore to the mounting medium of the microscopic preparation. The overall fluorescent background produced in this way, enabled calibration of local fluorescence intensities in different parts of one fluorophotographic negative, and also of the intensities in different negatives taken from one microscopic preparation. The validity of this approach was checked by comparing data obtained from several photographic negatives of the same quinacrine-stained metaphase, taken with different exposure times to imitate fluctuations in excitation illumination, after conversion of the scanning data into emission intensity values with an alogarithm based on the proposed theoretical relation. In another experiment, fluorescence emission intensities of Feulgen-stained chromosomes which had been measured with a cytofluorometer, were compared with results obtained by conversion of the scanning data measured in the fluorophotographic negatives of the same metaphases. Both types of experiment confirmed the applicability of the procedure described.

Chromosomes↗
PubMed1976

A theoretical study of concentration of profiles of primary cytochemical-enzyme reaction products in membrane-bound cell organelles and its application to lysosomal acid phosphatase.

A numerical method was developed for computing the steady-state concentration gradient of a diffusible enzyme reaction product in a membrane-limited compartment of a simplified theoretical cell model. In cytochemical enzyme reactions proceeding according to the metal-capture principle, the local concentration of the primary reaction product is an important factor in the onset of the precipitation process and in the distribution of the final reaction product. The following variables were incorporated into the model: enzyme activity, substrate concentration, Km, diffusion coefficient of substrate and product, particle radius and cell radius. The method was applied to lysosomal acid phosphatase. Numerical values for the variables were estimated from experimental data in the literature. The results show that the calculated phosphate concentrations inside lysosomes are several orders of magnitude lower than the critical concentrations for efficient phosphate capture found in a previous experimental model study. Reasons for this apparent discrepancy are discussed.

Acid Phosphatase↗
PubMed1976

Prospects for microscopical cytochemistry.

This review is devoted mainly to an evaluation of the status of microscopical cytochemistry seen as a discipline aiming at both the localization and the quantification of molecular processes in cells. Its relationships to ultramicrochemistry, as well as, in a broader sense, to biochemistry and cell biology, are discussed from both the historical and the methodological points of view. Recent developments in quantitative cytophysical techniques, such as automated cytophotometry using microscopes fitted with flying spot systems, TV cameras, or scanning stages, and the development of rapid flow cytometers are discussed. Analytical electron microscopy is touched upon too. The main part of the review is devoted to recent trends that strengthen the analytical basis of cytochemical staining methods. The special character of staining procedures as a kind of matrix chemistry is discussed and the potentialities of the use of matrix-incorporated compounds for the fundamental study and calibration of microscopical staining procedures are elaborated. Parallel developments in the theory and practice of matrix chemistry in biochemistry are stressed. Growing interrelations between microscopical cytochemistry and related fields of investigation, such as the controlled fragmentation of cells, and methods like ultramicroanalysis of individual cells are indicated.

Chemistry↗
PubMed1975

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.

Acetates↗