Search PubMed⌕ Search

Biomedical subjects

J F Leary

Publications and source records attributed to J F Leary.

At least 37 records · Page 2Linked to original sources
PubMed1988

Flow cytometric analysis of cell-surface binding elements for fibronectin on mouse lung cell isolates.

Fresh lung cell isolates from LAF1 mice were examined for the presence of fibronectin-binding elements using flow cytometric analysis. Thoroughly perfused lungs from adult male mice were dissociated using an elastase-trypsin digestion, gentle pipetting and filtering. The resulting heterogeneous cell suspension was incubated with fibronectin coated 0.5 micron fluorescent beads. Subsequent flow cytometric analysis indicated the presence of two species of specific fibronectin-binding populations; one of higher binding affinity which can be blocked with exogenous plasma fibronectin and one of lower binding affinity. We tentatively identify the lower affinity binding element with the fibronectin adhesion receptor and the higher affinity element with the putative matrix assembly receptor.

Animals↗
PubMed1987

Surface glycoproteins of differentiating neuroblastoma cells analyzed by lectin binding and flow cytometry.

Cell surface glycoproteins of mitotic neuroblastoma cells and cells differentiated by prostaglandin cyclic adenosine monophosphate treatment were quantified by flow cytometric analysis and specific fluorescent lectins. No differences in fluorescent lectin binding were seen between suspensions of mitotically active and differentiated N2AB-1 cells following exposure to either fluorescein (FL)-labeled soy bean agglutinin (FL-SBA) specific for N acetyl galactosamine or FL-concanavalin A (FL-CON A) which binds to mannose residues. These lectins, however, were shown to bind specifically to these cells as revealed by competitive blocking studies with hapten sugars. When FL Ulex europaeus (FL-UEA) specific for fucose was reacted with control or differentiated cells, no binding was seen even with an increased dose of lectin before or after enzyme treatment. However, differentiated N2AB-1 cells, reacted with FL-wheat germ agglutinin (FL-WGA) specific for N acetyl glucosamine, bound more FL-WGA than that seen for control cultures. Furthermore, specific sites for FL-WGA were shown to be saturable and were lost upon pretreatment of cells with neuraminidase. Neuraminidase pretreatment revealed masked sites for FL-CON A and FL-SBA since binding was increased at least twofold for these lectins on mitotic and differentiated cells. These data indicate that single cell measurements of surface glycoproteins can be made on living neural cells and that differentiation induces an increase in cell surface N-acetyl glucosamine residues.

Acetylgalactosamine↗
PubMed1987

Multipotent human hematopoietic cell line K562: lineage-specific constitutive and inducible antigens.

K562 cells have been reported to display a variety of non-erythroid properties. Using 28 lineage-specific monoclonal antibodies, we analysed which antigens are present spontaneously and which are inducible by a variety of agents. The data suggest that (1) antigens of a given lineage are preferentially responsive to certain inducers, e.g. megakaryocytic antigens to phorbol ester, and (2) a given inducer may influence antigens of different lineages in opposite directions, e.g. phorbol dibutyrate, not only induces megakaryocytic antigens, but also decreases granulocyte and erythroid antigens. We conclude that the K562 cell, despite its malignant origin, retains some capacity for expression of alternative programs of differentiation, a characteristic of the normal multipotent hematopoietic stem cell.

Antigens↗
PubMed1987

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2.

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.

Animals↗
PubMed1986

A quantitative study of the development of type II pneumocytes in fetal lung.

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II pneumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine-3R and by their intensity of low-angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2-d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low-angle light scatter and phosphine-3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24-d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P less than .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.

Age Factors↗
PubMed1986

Tetanus toxin binding to neuroblastoma cells differentiated by antimitotic agents.

We have examined the binding of tetanus toxin (TT) to surface receptors of neuroblastoma cells by flow cytometry following chemically induced differentiation. Cells were treated with mitomycin C, bromodeoxyuridine, prostaglandin E1 or cyclic adenosine monophosphate at different doses, alone or in combination for 4 days. Cells extended long neurites within 24 h in the presence of prostaglandin/cyclic AMP or mitomycin/bromodeoxyuridine treatment while single-drug treatment was less efficient in morphological differentiation of these cells. Cells exposed to the drug combinations stopped growing after 3 days while flow-cytometric analysis of DNA levels of each cell stained with propidium iodide indicated that at least 60% of these cells were arrested in phase G0/G1 of the cell cycle. Drug-treated cultures were stained for TT binding by immunofluorescence of cells in suspension and analyzed by flow cytometry. Chemically differentiated N2AB-1 cells were shown to bind significantly more TT than control cultures. Receptors for TT could be saturated by increasing doses of TT and differentiated cells bound twice as much toxin at saturation as did control cells. Immunofluorescence of TT binding to monolayers revealed staining in a stippled fashion along all neurites and cell bodies. These data support the concept that drugs which stimulate differentiation of neuroblastoma cells as determined by morphological and cell-cycle criteria also increase the presence of ganglioside receptors on the cell surface available for toxin binding.

Alprostadil↗
PubMed1985

Flow cytometric detection of lymphocyte alterations in Huntington's disease.

Several recent reports indicate that patients with Huntington's Disease (HD) may manifest membrane abnormalities in a wide variety of cells including peripheral blood lymphocytes. In this study, flow cytometry is used in conjunction with the fluorescent membrane probe, 8-anilino-1-naphthalene sulfonate (ANS), to examine peripheral blood lymphocytes from 16 HD patients and 14 age- and diet-matched control subjects. Increased ANS fluorescence intensity of lymphocytes (p less than 0.02) was found in HD patients as compared to control subjects. These differences are masked when the mean fluorescence of the total leukocyte population is measured, possibly explaining conflicting data of other investigators. These observed differences in ANS fluorescence intensity between HD patients and control subjects support the concept of a gene defect which may be expressed as membrane alterations in non-neural as well as neural cells. The selective alterations of lymphocytes may also reflect altered immunological activity reported in HD.

Adult↗
PubMed1985

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells.

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.

Cell Cycle↗
PubMed1985

Regulation of K562 cell transferrin receptors by exogenous iron.

Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.

Animals↗
PubMed1985

Flow cytometric analysis of tetanus toxin binding to neuroblastoma cells.

A neuroblastoma cell line was assessed for its capacity to bind tetanus toxin (TT) by using immunofluorescence and flow cytometry to analyze cells on a single cell basis. A clone of Neuro 2a, N2AB-1, was shown to bind variable amounts of TT per cell and this binding could be saturated by increasing doses of the toxin. Toxin binding was specific for neuronal cells, as the non-neuronal cell line, C6 glioma, bound negligible amounts of toxin. Variability of immunofluorescence staining was due in part to the increase in size of N2AB-1 cells as they progress through the cell cycle as measured by cell surface densities of toxin binding and DNA levels by propidium iodide (PI) staining. When N2AB-1 cells were treated with exogenous gangliosides for 24 h, cells were induced to sprout neurites and cell growth was inhibited. Analysis of DNA histograms indicated that ganglioside treatment caused more cells to appear in G0G1 of the cell cycle than that seen for untreated controls. Upon cytometric analysis of TT binding to ganglioside treated cells, it was apparent that treatment stimulated all cells to bind TT in larger amounts per cell than that seen with untreated N2AB-1 cells. These data suggest that TT binding and, therefore, toxin receptors are constant in density throughout the cell cycle of these neuroblastoma cells and that exogenous gangliosides can cause differentiation followed by increased toxin binding.

Animals↗
PubMed1984

Rare-event analysis methods for detection of fetal red blood cells in maternal blood.

Methods for analyzing very small subpopulations of cells ("rare-event analysis methods") are of interest in many areas of biology. In this paper we demonstrate the ability to detect one Rh-positive erythrocyte per 100,000 Rh-negative erythrocytes. This is the level needed to detect human fetal red blood cells in the maternal circulation in the case of fetal-maternal Rh-incompatibility. Erythrocytes stained with fluorescent immunobeads are more than 100 times as bright as the same cells stained by indirect immunofluorescence (or almost 40 times as bright as cells stained by the biotin-avidin technique). More importantly there is greater than a sevenfold increase in signal-to-noise (S/N) ratio, which proves critical in detecting very low frequency ("rare event") cell subpopulations. Although equal mixtures of positive and negative cells can easily be detected, positives cannot be easily distinguished from negatives when they are present in relative frequencies of 1:1000 or less as measured by either indirect immunofluorescence or biotin-fluorescent avidin labeling of D antigens. However, use of fluorescent immunobeads and high-speed flow cytometry allows detection of subpopulations of the same cells at relative frequencies of less than 1:100,000. Based on calibrations using free-fluorescent immunobeads, the average Rh-positive cell (when present in a relative frequency of 1:100,000) labeled with 36 +/- 19 fluorescent immunobeads. Rh-positive cells of relative frequencies 1:10,000 and 1:100,000 can be detected above background (Rh-negative red blood cells treated with the same amount of fluorescent immunobeads). Using defined mixtures of Rh-positive and negative cells, the expected numbers of positive cells were observed over a range of 1:100 to 1:100,000 relative Rh-positive cell frequencies. These methods should prove of general use in applications where original frequency information about rare cell subpopulations is important and would be lost if enrichment procedures were to be used.

Centrifugation, Density Gradient↗
PubMed1984

Somatic cell hybridization of human tumor samples.

Human intraspecific hybrids were formed between tumor cells isolated from both primary and metastatic tumors and a tissue culture adapted cell line, D98OR, a HeLa derivative which is thioguanine and ouabain resistant. Five different tumor types in all were attempted: renal cell carcinoma, colon adenocarcinoma, melanoma, chrondrosarcoma, and hepatocarcinoma. The tumor tissue was either (1) immediately dissociated and fused, or (2) frozen and later thawed, dissociated, and fused. Two different PEG concentrations were used. The results reported here demonstrate that: (1) hybrid tumor cell lines can be made from several types of cancer, (2) unfrozen tumor tissue fused with D98OR by exposure to 50% PEG appears optimal, (3) chromosome loss, as determined by flow cytometry studies of hybrid DNA content, is minimal, and (4) hybrids have characteristics consistent with derivation from tumor cells rather than derivation from the nonmalignant cells of a tumor.

Cell Fusion↗
PubMed1982

Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry.

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single-cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Since large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.

Alpharetrovirus↗
PubMed1982

Adsorption of Rous sarcoma virus to genetically susceptible and resistant chicken cells studied by laser flow cytometry.

Quantitative binding of Rous sarcoma virus (RSV) of different antigenic subgroups to chicken cells was examined by using a laser flow cytometer/cell sorter. RSV of subgroups A, C, and E, labeled with the fluorescent membrane probe rhodamine-18, bound 2 to 10 times more to genetically susceptible chicken embryo fibroblasts than to resistant cells, as measured by flow cytometry on a single-cell basis. This suggested that susceptible cells possess both specific and nonspecific receptors for virus adsorption, whereas resistant cells bind virus only by means of nonspecific sites. Polybrene at low concentration increased eightfold the binding of virus. Higher levels of Polybrene inhibited adsorption. Cell binding sites were saturable, and attachment of labeled virus could be partially blocked by preexposure of cells to unlabeled RSV. Virus surface glycoproteins played an important role in adsorption, since their removal with bromelain decreased binding of virus to susceptible cells. Maximal binding of RSV to both susceptible and resistant cells occurred within 10 min, although the level of binding was up to 10-fold higher for susceptible cells. Binding to all cell types showed a broad distribution. This implies that there are considerable differences in the number of virions bound per cell.

Adsorption↗
PubMed1982

Isolation of type II pneumocytes by laser flow cytometry.

The isolation of a pure population of type II pneumocytes from adult rabbit lung by the technique of multiparameter laser flow cytometry is reported here. Lipophilic phosphine-3R was used to stain cell dissociated from lungs by elastase and trypsin. Type II cells were then sorted by simultaneous measurement of high phosphine-3R fluorescent staining of lamellar bodies and intermediate intensity of low-angle light scatter. We were able to obtain an essentially pure population (98%) at sorting rates of 5 X 10(5) lamellar-body-containing cells per hour. The cell sorter technique offers an attractive alternative to the density gradient isolation of type II cells, particularly in terms of detailed population analysis. In addition, refinements in cell sorter methods offer the potential to discriminate differentiating type II cells with few lamellar bodies that do not segregate on the basis of buoyant density.

Acridines↗
PubMed1980

Rapid, quantitative analysis of cell cycle stages of cold-sensitive derivatives of the Chinese hamster cell line CHO-K1.

Cell cycle parameters of two cold-sensitive Chinese hamster cell lines have been determined by flow cytometric analysis of cell populations stained with the DNA specific fluorochrome mithramycin. The most striking finding is a depletion of S phase cells, detectable as early as 12 h after a shift to the nonpermissive temperature of 30.0 degrees C and complete by 24 h following the shift. There is a substantial increase in the proportion of cells in the G1 phase of the cell cycle compared to wild type cells under identical conditions but, surprisingly, the proportion of cells having a G2/M DNA content is quite similar in the two populations. The proportion of tetraploid cells present in these populations is not sufficient to account for this observation. Reversibility of the cold-induced block was tested by returning cells held for three days at the nonpermissive temperature to the permissive temperature. Cells having a G1 content of DNA do reenter the S phase, beginning approximately 8 h after a return to the permissive temperature.

Animals↗
PubMed1979

Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells.

In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.

Animals↗