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At least 415 records · Page 23Linked to original sources
PubMed1997

Functional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0 /G1 phase of the cell cycle.

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34(+) cells isolated in G0 (G0CD34(+) cells) than in those residing in G1 (G1CD34(+) cells). However, as MPB CD34(+) cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34(+) cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34(+) cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34(+) cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34(+) cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2(dim) population whereby LTHC-IC frequency was higher for CD34(+) cells reselected in G0 after in vitro division than for CD34(+) cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2(bright) CD34(+) cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

Antigens, CD34↗
PubMed1976

[A case of lupus pancreatitis (?) in systemic lupus erythematosus].

The cause of death of a 52 year old female suffering from disseminated lupus erythematosus was acute (steroide-induced?) pancreatitis. Histologically in the pancreas spontan nucleophagocytosis, leukophagocytosis, erythrophagocytosis, presence of haemtoxylin-body-like structures could be revealed by HE, methyl-green-pyronin staining and by Feulgen reaction.

Adrenal Cortex Hormones↗
PubMed1976

[Immunological response of mice to diphtheria toxoid].

Following the determination of immunological response in mice, by titration, 1.5 Lf diphtheria toxoid was found as an optimal plasma cell activator. Groups of mice were subcutaneously injected with 1.5 Lf diphteria toxoid and on 2, 3, 4, 6, 10, 15 and 25th days of injection mice were killed and their blood, spleen, thymus, lymph nodes and lungs preserved. Section from these specimens were stained by methyl-green, pyronine and toluidine. Lymphocytes, plasma cells, and mast cells were counted in 10-20 fields in each preparation. Mature plasma cell ratio was calculated against lymphocyte counts. Mature plasma cell and mast cell counts and ratios of test mice were found increased when compared with that of normal control mice. The difference in thymus, spleen and lungs was found to be insignificant, however it was significant, p less than 0.05 in lymph. nodes. Maximum cell counts were seen at ten days. The sera of the test mice were also examined for humoral antibodies with precipitation and Römer's skin test. on 2, 3, 4, 6 and 10th days no antitoxin could be demonstrated in the sera of the test mice in controls either. In the sera taken on 15 and 25th days antitoxin was detected.

Animals↗
PubMed1998

Chronic nonspecific endometritis.

To assess the proliferative activity of glands and stroma in nonspecific chronic endometritis (NCE), we evaluated the plasma and morphologic features. We examined 25 endometrial sections that were coded as inactive for the morphologic features of NCE other than plasma cells and 25 proliferative endometria (PE) as controls. Furthermore, the sections were stained with methyl green pyronin (MGP) to demonstrate plasma cells and proliferative cell nuclear antigen (PCNA) for proliferative activity. The number of plasma cells and the grade of proliferative activity were determined by a semiquantitative scale. The results were compared by using the Mann-Whitney-U-test. Of the 25 inactive endometria cases, 11 were NCE and 14 were either early proliferative endometria (seven cases), lower uterine segments (five cases), or under hormone effect (two cases). The number of plasma cells in NCE was significantly higher than in PE. However, there were cases of NCE without plasma cells and cases of PE containing plasma cells. Proliferative activity was significantly lower in NCE cases than in the PE group (p < 0.05). The diagnosis of NCE should rely more on morphologic abnormalities than on plasma cell criteria. Identification of plasma cells may be useful for diagnosis.

Cell Division↗
PubMed1998

Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0.

Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.

Adult↗
PubMed1995

Nucleic acid dyes for detection of apoptosis in live cells.

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy-1 dim thymocytes were included in the dyc dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin. However, the dyes SYTO-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser.

Acridine Orange↗
PubMed1981

Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity.

A temperature-sensitive mutant of E. coli, WG24, was studied with respect to its sensitivity to photodynamic action, its capacity to perform host controlled reactivation, and its sensitivity to transduction at elevated temperatures. Mutant cells are much more sensitive than wild type cells to photodynamic action by thiopyronine and visible light at elevated temperatures. As well defined rec mutants, WG24 cells are less able to reactivate UV irradiated lambda c phages at elevated temperatures, while their ability to repair T1 phages is less impaired. Mutant cells cannot be transduced to T6 resistance at a detectable rate at elevated temperature. It is concluded, therefore, that some rec gene carries a ts mutation in this mutant.

Coliphages↗
PubMed1985

Altered orientation of glycosaminoglycans and cellular changes in the tibial cartilage in the first two weeks of experimental canine osteoarthritis.

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

Alcian Blue↗
PubMed1996

Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.

Visualization of DNA in electrophoretic gels typically requires UV radiation and the fluorescent dye ethidium bromide. Alternatively, we report here that by inclusion of visible dyes in standard agarose gels, DNA bands are observable in ambient light as they are separating. Such bands can be directly recovered from gels (approximately 50% yield) and used in standard enzymatic reactions (ligation, endonucleolytic cleavage, random labeling, PCR, and cycle-sequencing) without purification. Of 14 common commercially available stains that could visualize fractionating DNA, Nile blue was chosen for more extensive analysis as it gave the sharpest and most persistent bands and is not known to be toxic. Bands containing greater than 40 ng DNA could be detected by direct visual inspection of gels during electrophoresis. Drying the gels increased sensitivity to 4 ng. We describe relevant molecular features of these dyes and detail simple assays that may be employed to find other useful, and perhaps superior, dyes. This method also lends itself to situations in which easy visualization and convenience of DNA electrophoresis are important, such as classroom demonstrations.

Blotting, Southern↗
PubMed1980

A rapid method for measuring drug enrichment in epidermis.

A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2-10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12-36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude. The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm3. Using this value, tissue weight can be translated into volume and concentraton changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest. The enrichment of five substances in human epidermis was measured: alpha-estradiol, thiopyronincce, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10-500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronin and 8-MOP, reversible binding is indicated with dissociation constants of 10(-7) M and 10(-5) M, respectively, while partitioning distribution could account for the behavior of 5-MOP and alpha-estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.

Binding Sites↗
PubMed1991

Investigation of some antimicrobial procedures on the in vitro development of early murine embryos aimed toward developing methods for the disinfection of mammalian embryos prior to transfer.

Eight-cell, zona pellucida-intact mouse embryos were exposed to the following substances or procedures that have been reported to have germicidal effects to determine if the embryos would survive and develop under in vitro conditions: the photosensitive substances hematoporphyrin, hematoporphyrin derivative, 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and thiopyronine; the enzymes lipase (0.5%), phospholipase C (2 U/ml), chymotrypsin (0.5%), and trypsin (0.5%); pH 5.0; and helium/neon laser light, visible light, ultraviolet A light, and ultraviolet C light. Under the conditions used, embryos were not adversely affected by hematoporphyrin and/or helium/neon laser light; methoxypsoralen and/or ultraviolet A light; lipase; trypsin; pH 5.0 for 20 min; and visible light. Variable results were obtained from hematoporphyrin derivative with laser light. Thiopyronine, trimethylpsoralen in combination with ultraviolet A light, and ultraviolet C light killed embryos, and chymotrypsin and phospholipase C were harmful at 10- and 15-min exposure times, respectively.

Animals↗
PubMed1985

Thiopyronine and 8-methoxypsoralen sensitized photodynamic effect on DNA synthesis in yeast.

The synthesis of DNA in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine (TP) and visible light or with 8-methoxypsoralen (8-MOP) and UVA light. DNA synthesis was inhibited after photodynamic treatment with 8-MOP but not after photodynamic treatment with TP. This result is further evidence that the photodynamic effect with TP does not attack nuclear DNA in eucaryotic cells.

DNA, Fungal↗
PubMed1978

Induction of mutations by photodynamic action of thiopyronine in Saccharomyces cerevisiae.

The induction of cytoplasmic and nuclear mutations by the photodynamic action of thiopyronine is demonstrated in a haploid strain of Saccharomyces cerevisiae that has been isolated as a photodynamic sensitive mutant. No significant increase in corresponding mutation frequencies could be observed in a strain resistant to photodynamic inactivation by thiopyronine.

Cell Nucleus↗
PubMed1981

UV-induced reactivation and mutagenesis of lambda-phages after treatment with 8-methoxypsoralen or thiopyronine and light.

Lesions, which were produced on lambda-phages DNA by the photosensitization effect of 8-methoxypsoralen (8-MOP) can be repaired by UV-induced repair systems (W-reactivation) in Escherichia coli wild type host cells. By optimum induction of the repair system, about 45% of the 8-MOP lesions are repaired. The survival of lambda-phages inactivated by the photodynamic action of thiopyronine (TP) is only negligibly increased by the same UV-induced repair systems. However, the frequencies of clear plaque mutations of 8-MOP treated as well as TP treated lambda-phages increase in similar fashion if the host cells of wild type have been irradiated with UV. These results show the different capacities of induced repair systems in repairing different types of lesions. They also suggest that some types of base damages are repaired more error-prone than other DNA-lesions.

Bacteriophage lambda↗