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Biomedical subjects

J Tas

Publications and source records attributed to J Tas.

At least 19 recordsLinked to original sources

Effect of a Trichinella spiralis infection on the distribution of mast cell precursors in tissues of thymus-bearing and non-thymus-bearing (nude) mice determined by an in vitro assay.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.

Animals↗

DNA and nuclear protein measurement in isolated nuclei of human endometrium.

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.

Adult↗

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium.

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.

Adenocarcinoma↗

Detection of metabolic changes in hepatocytes by quantitative cytochemistry.

Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.

Alanine Transaminase↗

A visible DNA-protein stain: Feulgen-Pararosanilin(SO2) Light Green.

Feulgen-Pararosanilin(SO2) Light Green, a DNA-protein stain, is described that is suited both for visual analysis and quantitative cytochemical measurement. The stain has been applied on cervical cells and quantitative aspects have been studied on chicken erythrocyte and rat liver nuclei. In comparison to single staining, the Feulgen-Pararosanilin(SO2) DNA content of the nuclei remains unaltered after application of the combined staining. The Light Green protein content is reduced considerably, however, dependent on the degree of chromatin condensation in the nucleus.

Animals↗

The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA.

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.

Animals↗

In vitro studies on mast cell proliferation in N. brasiliensis infection.

We have previously shown that mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature in rat bone marrow cultures stimulated with factors from antigen or mitogen-activated T lymphocytes. Here we have used this system to explore the MMC hyperplasia which occurs in infections with gastrointestinal nematode parasites. Lymphocytes producing MMC-growth factor were present from day 10 onwards in N. brasiliensis-infected rats and mesenteric lymph nodes (MLN) were the major source of activated lymphocytes. When different tissues of normal rats were cultured in the presence of conditioned medium by far the greatest proliferation of MMC occurred in bone marrow, indicating an origin of MMC from haemopoietic precursors. Cultures of infected rat bone marrow yielded considerably greater numbers of MMC than cultures of normal rat bone marrow and experiments using semisolid culture media indicated that N. brasiliensis infection causes an increase in the frequency of MMC progenitors in the bone marrow. A scheme is put forward for the sequence of events occurring in vivo based on the results of these and other published experiments. The reasons for the restricted in vivo localization of MMC to the mucous membranes and associated lymph nodes is discussed. Finally we give the results of microspectrophotometric analysis which has shown that the cultured mast cell contain a non-heparin proteoglycan, thus adding a further feature to the list of MMC-like properties of these cells.

Animals↗

Glucose-6-phosphate dehydrogenase activity in individual rat hepatocytes of different ploidy classes. I. Developments during postnatal growth.

The glucose-6-phosphate dehydrogenase (G6PDH) activity of isolated male rat hepatocytes has been investigated in relationship to the ploidy classes of the cells during the first 20 weeks of postnatal growth. The G6PDH activity in the individual cells was measured with an improved quantitative cytochemical method. The data obtained showed that throughout the whole period of postnatal growth there existed a proportional relationship between the genome copies per cell and the amount of G6PDH activity per cell for binuclear diploid (BD), mononuclear tetraploid (MT) and binuclear tetraploid (BT) cells but not for mononuclear diploid (MD) cells. In the MD cells, which are the stem cells of the liver parenchyma, the activity measured was 1.5 times higher than expected. Furthermore, during postnatal growth, the G6PDH activity per hepatocyte was low at the age of 2 weeks, increased somewhat after weaning (5 weeks) and then more dramatically after 8 weeks to reach a maximum between 12 and 16 weeks. This development occurred in MT and BT cells at an earlier age than in MD and BD cells, in which the increase in enzyme activity followed some 3 weeks later. Castration of the rats before puberty did not influence the development of the amount of G6PDH activity per cell of any of the ploidy classes.

Animals↗

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects.

Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1 M MgCl2, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacke purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl2 staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl2 were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.

Animals↗

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy.

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.

Animals↗

Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.

With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into units of enzyme activity. This conversion enabled cytochemical data to be compared directly with biochemical values. The conversion was applied to the cytochemical estimation of glucose-6-phosphate dehydrogenase activity in isolated rat hepatocytes, mouse oocytes, rabbit thymocytes, human granulocytes and human fibroblasts. Several control procedures were performed to confirm the admissibility of this conversion, such as: the estimation of the absorption characteristics of the formazans of tetranitro blue tetrazolium both in solution and precipitated in biological specimens; the linearity of the relationship between the increase of absorbance and incubation time; and the effect of different incubation conditions on the amount of specific formazan production.

Animals↗

A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier.

A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cell is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used.

Acrylic Resins↗

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a "fixation" in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.

Erythrocytes↗

On the DNA content and ploidy of trypanosomes.

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture from were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1,221-246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.

Animals↗

The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.

Acrylic Resins↗

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Animals↗