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Biomedical subjects

H B Steen

Publications and source records attributed to H B Steen.

At least 73 records · Page 4Linked to original sources
PubMed1986

Fluorescence spectra of cells stained with a DNA-specific dye, measured by flow cytometry.

Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.

Animals↗
PubMed1986

Binding of Hoechst 33258 to chromatin in situ.

The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 10(7) M-1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 less than or equal to bound dye/phosphate less than or equal to 0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (greater than or equal to 15 degrees) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (greater than or equal to 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient approximately 2) and the apparent binding constant was 2-3 X 10(5) M-1 as determined from quenching and precipitation data.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Simultaneous separate detection of low angle and large angle light scattering in an arc lamp-based flow cytometer.

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp-based flow cytometer with epi-illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2 degrees and 15 degrees, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser-based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25-micron diameter. A potential application may be differentiation of bacteria.

Animals↗
PubMed1986

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.

Chloramphenicol↗
PubMed1986

Flow cytometry of bacteria: cell cycle kinetics and effects of antibiotics.

Flow cytometric determination of the DNA and protein content of E. coli has been carried out by means of a microscope-based flow cytophotometer with a high pressure arc lamp excitation ligh source. Fluorescence (DNA)/light scatter (total cell protein) dual parameter histograms with a resolution cv of 5% were obtained for cells labeled with a combination of mithramycin and ethidium bromide. Histograms of E. coli in rapid and slow exponential growth are presented to exemplify how the cell cycle kinetics of bacteria can be studied in much more detail than has been possible by other methods. Significant effects of chloramphenicol and penicillin on the cell cycle distribution and cell numbers of E. coli cultures were evident after one hour of culture. The data provided information on which parts of the cell cycle and what types of processes were affected by the drug. It appears that flow cytometry may become a valuable tool in studies of the cell cycle of bacteria as well as in clinical drug testing.

Anti-Bacterial Agents↗
PubMed1985

Neither adriamycin nor actinomycin D displaces Tb3+ from DNA.

The decay of fluorescence of Tb3+ bound to DNA was measured in the absence and presence of adriamycin and actinomycin D. The decay for Tb3+ bound to DNA was mainly exponential (lifetime: tau = 0.96 ms). In the presence of adriamycin or actinomycin D, the Tb3+ fluorescence decayed much faster, indicating that excitation energy was transferred from Tb3+ to the drugs. Extrapolation of the decay curves to zero time showed that the number of strongly emitting, DNA-bound terbium ions was not reduced by the presence of adriamycin or actinomycin D. Hence, these drugs do not seem to displace Tb3+ bound to DNA.

Binding, Competitive↗
PubMed1985

Differential of light-scattering detection in an arc-lamp-based epi-illumination flow cytometer.

Light-scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc-lamp-based flow cytometer (AL-FCM). The results were compared with those obtained with a conventional, laser-based flow cytometer (L-FCM) with forward scattering (2-20 degrees) and scattering at right angles. Measuring with the AL-FCM in the angle range upward from 13 degrees, the relative light-scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right-angle scattering in the L-FCM. With a range of scattering angles upward from 2 degrees the AL-FCM yielded histograms similar although not identical to that of the forward-scattering detector in the L-FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.

Blood Cells↗
PubMed1985

Antibodies to surface IgM and IgD increase the expression of various class II antigens on human B cells.

Antibodies to surface IgM and IgD were found to induce increased expression of class II antigens on normal and neoplastic human B cells within 24 h of stimulation. Antigens associated with different class II sub-locus genes (DC, DR and SB) were all found to be increased as determined by monoclonal antibodies (Leu-10 and B 3/4 for DC, D 1/12 for DR and MHM4 for SB-associated antigens). The increased expression of class II antigens was selective as anti-immunoglobulins failed to increase expression of other surface antigens such as B1 and beta 2-microglobulin. The effect of anti-mu and anti-delta could be blocked specifically by corresponding myeloma proteins suggesting that antibodies to surface IgM and IgD, respectively, were responsible for the effect observed. Moreover, antibodies to another surface antigen (B1) failed to induce such changes. Increased class II antigen expression appeared to be dependent on protein synthesis, and early changes in ion fluxes, but could not be elicited by membrane depolarization as reported in murine systems.

Antibodies, Anti-Idiotypic↗
PubMed1985

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.

Chloramphenicol↗
PubMed1985

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.

Cell Division↗
PubMed1985

Multiple binding modes for Hoechst 33258 to DNA.

Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.

Benzimidazoles↗
PubMed1984

Epstein-Barr virus binding to virus-carrying cell lines is enhanced in the presence of C3 and C3d.

The relationship between the receptors for the Epstein-Barr virus (EBV) and the C3d fragment of complement was investigated at the molecular level. In the presence of cell-bound C3, virus binding was enhanced in EBV genome-carrying lines. An identical effect could be elicited by C3d at one-quarter the weight amount; C3b and methylamine-treated C3 had no effect on virus binding. The minimum concentration of C3 which produced significant enhancement was 25 micrograms/ml. Virus binding increases were observed only after 20 min of complement-cell co-incubation. The response was not noted with EBV-negative lines and was independent of virus strain assayed (B95-8 and P3HR-1). These studies suggest that the binding sites for the two moieties are distinct, although they both involve the same cell surface complex. The two receptors are believed to display cooperativity.

Cell Line↗
PubMed1984

Different fate of antibodies to surface IgM and IgD in germinal centre cell-associated lymphomas.

In all of four human germinal centre cell-associated B lymphomas carrying sIgM + sIgD F(ab')2 fragments of rabbit antibodies to human mu-chain were accumulated intracellularly, whereas the accumulation of antibodies to delta-chain was considerably less abundant. The accumulation of antibodies to light chain was intermediate between antibodies to mu- and delta-chain. These results could be explained by the following observations: (i) the difference in the level of reexpression of sIgM and sIgD and (ii) the discovery that antibodies to delta-chain bound to reexpressed sIgD were degraded at a higher rate than those bound to primary sIgD and at a higher rate than antibodies to mu-chain, whether bound to primary or reexpressed sIgM.

Absorption↗
PubMed1983

A microscope-based flow cytophotometer.

By means of a new flow chamber, a standard fluorescence microscope with Epi illumination and 100 W mercury arc excitation has been turned into a flow cytophotometer combining high resolution and sensitivity with simplicity of operation. In the flow chamber, cells are passed in a narrow stream through the microscope focus carried by a laminar flow of water running on the open surface of a cover glass which is coupled to the oil immersion microscope objective. Two spectral components of the fluorescence, for example, resulting from specific staining of two different cellular constituents with different dyes, can be measured simultaneously in separate channels so as to produce three-dimensional histograms. The scattered light of the cells is detected in dark field by a second microscope situated opposite the primary objective. Scattered light detection is integrating with regard to scattering angle from 0 degree to 90 degrees. Hence, diffraction pattern effects are eliminated and the light scatter signal is approximately proportional to cell dry weight. The Epi illumination, which implies that excitation and fluorescence collection are parfocal, greatly simplifies instrument adjustment, which is further facilitated by the fact that the cell stream can be viewed at high magnification. Cell measuring time is about 3 microseconds which implies a measuring rate of 3 x 10(3) cells/s at 1% coincidence rate. Sensitivity is sufficient for measuring the DNA content of bacteria (that is, approximately 5 x 10(-15) g/cell) with a coefficient of variance (CV) of about 6%. CV less than 1% is achieved for DNA histograms of mammalian cells. A 5 W argon laser as excitation source facilitates slit scan analysis and increases the sensitivity and measuring rate by one to two orders of magnitude.

Flow Cytometry↗
PubMed1983

Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology.

The DNA and protein content of individual Escherichia coli cells were measured at a rate of 10(4) cells per second with a sensitive microscope-based flow cytometer. DNA and protein were quantified by measuring the fluorescence from cells stained with a combination of the DNA-binding drugs Mithramycin and ethidium bromide and by scattered light, respectively. Separate experiments demonstrated that the light scatter signal was proportional to protein content. Dual parameter histograms (fluorescence/scattered light) of bacterial cultures gave detailed pictures of changes dependent upon the growth conditions and of the cell cycle kinetics. Effects of antibiotics could be readily detected and characterized after a few hours. The results demonstrate that flow cytometry is a promising method for application in experimental and clinical microbiology.

Bacterial Proteins↗
PubMed1983

Different responses elicited in vitro by antibodies to IgM and IgD in cells from a surface IgM + D-positive human follicular lymphoma.

The effect of anti-Igs in combination with the tumour promotor TPA on DNA and Ig synthesis in a human follicular germinal centre cell lymphoma carrying sIgM and sIgD was investigated. The lymphoma cells responded to both anti-mu and anti-delta chains and to anti-lambda light chains by DNA synthesis, as measured by methyl-(3H)-thymidine incorporation. Flow cytofluorometric measurements, however, showed that only anti-mu chains induced marked increase in cytoplasmic Ig content. This result suggests that in certain B-cell subsets the signals elicited via sIgM and sIgD are different.

Antibodies, Anti-Idiotypic↗
PubMed1983

Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry.

The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.

Bacterial Proteins↗