PubMed Health⌕ Search

Biomedical subjects

R Habbersett

Publications and source records attributed to R Habbersett.

12 recordsLinked to original sources
PubMed1997

Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells.

The effects of arachidonic acid (AA) and gamma-linolenic acid (GLA) on cell cycle progression and apoptosis induction, using flow cytometry, were compared on normal human skin fibroblasts, strain HSF43 with wild type (wt) p53, large T antigen transformed HSF43 cells (line E8T4) with non functional p53, and on two lymphoblast cell lines, TK6 with wt p53 and WTK1 with mutant p53. AA and GLA caused similar dose (50, 75 and 100 microg/ml AA and GLA) and time dependent (24 and 48 h) induction of apoptosis in each cell line. The degrees of the response of the four cell lines were, however, different. The normal HSF43 cells were most resistant against apoptosis induction and the WTK1 cells most susceptible. The apoptosis induction appeared to be independent of functional p53. Cell cycle progression was also similarly affected by AA and GLA in the two cell types. In the fibroblast type cells (HSF43 and E8T4) S- and G2/M-phase arrests were evident after 48 h exposure to AA and GLA, and in the lymphoblast cell lines (TK6 and WTK1) the cells were arrested in the G1-phase.

Apoptosis↗
PubMed1997

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Chromosomes, Human↗
PubMed1996

Alterations in the progression of cells through the cell cycle after exposure to alpha particles or gamma rays.

A G1-phase delay after exposure to alpha particles has not been report ed previously, perhaps because immortalized cell lines or cell lines from tumor cells were used in past studies. Therefore, we compared the effects of alpha particles (0.19 or 0.57 Gy) and approximately equitoxic doses of gamma rays (2 or 4 Gy) on progression of cells through the cell cycle in normal human skin fibroblasts. Cell cycle analyses were performed using flow cytometry by measuring incorporation of bromodeoxyuridine (BrdUrd) in each phase of the cell cycle up to 44 h after irradiation. We observed an alpha-particle-induced G1-phase delay in human skin fibroblasts even at the lowest dose, 0.19 Gy. At equitoxic doses, more pronounced and persistent G1-phase delays and arrests were observed in gamma-irradiated cultures in that increased fractions of the G1-phase cells remained BrdUrd- over the course of the study after gamma-ray exposure compared to cells exposed to alpha particles. In addition, G1-phase cells that became BrdUrd+ after gamma irradiation re-arrested in G1 phase, whereas BrdUrd+ G1-phase cells in alpha-particle-irradiated cultures continued cycling. In contrast, comparable percentages of cells were delayed in G2 phase after either alpha-particle or gamma irradiation. Both gamma and alpha-particle irradiation caused increases in cellular p53 and p2lCip1 shortly after the exposures, which suggests that the G1-phase delay that occurs in response to alpha-particle irradiation is dependent on p53 like the initial G1-phase delay induced by gamma rays.

Alpha Particles↗
PubMed1995

Simultaneous dual-frequency phase-sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome-labeled cells and particles.

In frequency-domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase-shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring lifetimes at multiple frequencies. In this article we explore the requirements and experimental design considerations for making such measurements in flow. We report a phase-sensitive flow cytometric method that allows one to probe the excited state-lifetimes of labeled cells by using multiple simultaneous modulation frequencies. Application of this method is demonstrated by measuring fluorescence lifetimes of labeled cells at two frequencies simultaneously, using a continuous-wave, dual-frequency modulated excitation in flow. The dual-frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell-by-cell basis in real time. Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level.

Animals↗
PubMed1995

A rapid mix flow cytometer with subsecond kinetic resolution.

Kinetic approaches are valuable tools for mechanistic studies of cell function. Flow cytometry is well suited to make sensitive kinetic measurements, but the time required to deliver mixed samples to the point of measurement (10-20 s in a conventional cytometer) limits analysis of rapidly occurring events. To address this limitation, we adapted a syringe-based stopped-flow rapid mixing device to a modified commercial flow cytometer to achieve mixing and measurement of sample in under 1 s. Because such screw-driven mixers are designed to deliver fluid at rates of microliters per millisecond and cytometers accept samples at microliters per second, the syringe mixer was modified with a screw to allow sample delivery at rates as low as 1.8 microliters/s. A custom-made nozzle holder featuring a fast-acting three-way sample delivery valve and a 1.5- microliters dead volume was designed for a Becton Dickinson FACS stream-in-air flow nozzle. Syringe motors and valves are computer controlled, as is the start signal for an adjustable time ramp. A stable sample stream can be established within the sheath stream in less than 1 s, enabling fluorescence measurements of microspheres with coefficients of variation of approximately 5%. Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at times of under 300 ms. Efficient mixing of reagents is demonstrated by the iodide quenching of microspheres surface labeled with fluorescein isothiocyanate (FITC). The instrument is capable of quantitatively proportioning cells and reagent, thereby allowing precise control of reagent concentration and dilution. Rapid kinetic measurements of intact cells are demonstrated by FITC-formyl peptide binding to cell surface receptors.

Flow Cytometry↗
PubMed1994

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics.

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.

Amino Acid Sequence↗
PubMed1991

Expression of the c-fgr and hck protein-tyrosine kinases in acute myeloid leukemic blasts is associated with early commitment and differentiation events in the monocytic and granulocytic lineages.

Two members of the src proto-oncogene family of intracellular tyrosine kinases, c-fgr and hck, are selectively expressed in differentiated myeloid cells. To study the expression of these genes in acute myeloid leukemia (AML) and to determine the specific myeloid lineages and stages of myeloid differentiation at which the expression of these genes is acquired, we used a series of 79 cases of de novo AML as a differentiation model. The levels of c-fgr, hck, and c-fms (encoding the colony-stimulating factor-1 receptor) mRNA transcripts were correlated with the presence of specific cell surface antigens and the morphologic and cytochemical features in these AML blasts. Relatively undifferentiated leukemic myeloblasts with an HLA-DR, CD34, CD33, CD13+/- cell surface immunophenotype (French-American-British [FAB] M1 or M2) were characterized by a lack of c-fms and c-fgr expression, while low levels of c-fms and c-fgr could be detected in undifferentiated myeloblasts (FAB M1 or M2), which also expressed CD14 at low antigen density. The hck transcripts were either undetectable in these cells or were expressed at low levels. In contrast, only hck mRNA transcripts could be identified in blasts with progranulocytic morphology (FAB M3), while c-fms, c-fgr, and hck were all expressed at high levels in blasts with differentiated myelomonocytic or monocytic features (FAB M4 and M5). No c-fms, c-fgr, or hck transcripts were evident in leukemic cells of the erythroid lineage (FAB M6). When undifferentiated leukemic myeloblasts (HLA-DR, CD34, and CD33) were induced to differentiate in vitro to cells with monocytic characteristics, the expression of c-fms, c-fgr, and the CD14 cell surface antigen were induced to high levels, accompanied by the acquisition of hck and CD13 expression. In contrast, when HLA-DR, CD34, and CD33 blasts were induced to differentiate in vitro to cells with granulocytic characteristics, only hck and CD13 expression were induced. Our data suggest that the acquisition of c-fgr and/or hck expression is associated with early commitment and differentiation events in distinct myeloid lineages. Assessment of the expression of these kinases may provide a molecular tool to assign lineage in AML in conjunction with morphology, cytochemistry, and cell surface antigen expression.

Cell Differentiation↗
PubMed1990

Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry.

Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.

Animals↗
PubMed1982

B lymphocyte subpopulation defined by a rat monoclonal antibody, 14G8.

14G8 is monoclonal rat antibody that recognizes an antigen found on 30 to 40% of B lymphocytes from normal mice and on approximately 65% of B lymphocytes from mice with the xid-determined immune defect. 14G8+ B cells from normal mice resemble B cells from mice with the xid-determined defect in that the median amount of membrane IgM expressed per cell is much larger than that of 14G8- B cells. The frequency of 14G8+ cells is highest in neonatal mice (approximately 55% of all spleen cells) and falls with age to approximately 25% of all spleen cells in adult mice. Relatively few lymph node or bone marrow B cells express the antigen recognized by 14G8. 14G8 also reacts with 50% of resident peritoneal cells and with red blood cells. 14G8+ and 14G8- B cell preparations were obtained by fluorescence-activated cell sorting and by adherence to 14G8 coated dishes. 14G8+ cells responded with in vitro proliferation to both anti-mu and to LPS. Cell cycle analysis indicated that approximately 33% of these cells entered S phase in response to LPS and 38% in response to anti-mu. In contrast, 14G8- cells responded poorly to LPS (7% of cells entered S phase) although they showed good responses to anti-mu (40% of cells entered S phase). Thus, 14G8+ B cells, despite their similarity to B cells from mice with the xid defect, can proliferate to anti-mu, which B cells from defective mice fail to do. 14G8 provides a monoclonal antibody valuable in the description of functional B cell subpopulations.

Aging↗
PubMed1981

Flow microfluorometric analysis of H-2L expression.

The cell surface expression of H-2L, a major transplantation antigen, was compared by flow microfluorometry to the expression of products of H-2K and H-2D loci, using monoclonal antibodies. By this methodology, the ontogeny and tissue distribution of Ld antigens were found to be indistinguishable from those of the K and D antigens. In a reciprocal blocking assay, using fluorescein-labeled test reagents, it was shown that monoclonals anti-H-2.65 and anti-H-2.64 did not inhibit the binding of each other. These results suggest that the alloantigenic determinants H-2.64 and H-2.65 are located at distinct sites on Ld molecules. Quantitative comparisons using the fluorescein-labeled monoclonal reagents indicated that Ld molecules are expressed at 2- to 3-fold lower levels on the cell surface compared with K and D molecules. These findings give new credence to a "3-locus" model for the major histocompatibility complex of man and mouse, where H-2L and HLA-C share several homologies that are unique and distinguish them from the other histocompatibility loci.

Animals↗