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B R Ware

Publications and source records attributed to B R Ware.

At least 37 records · Page 2Linked to original sources
PubMed1981

Fluorescence photobleaching recovery in solutions of labeled actin.

We have demonstrated that the technique of fluorescence photobleaching recovery (FPR) can be used to examine the state of a single component in complex self-assembling macromolecular systems. Polymerization of actin, initiated by addition of salt or Mg+2 to a low-ionic-strength solution of G-actin, has been observed by sequential measurement of FPR with the aid of fluorescein-labeled actin. Solutions of actin which had been labeled using 5-iodoacetamido fluorescein (5-IAF) showed anomalous recovery of fluorescence above the initial value, which indicates a photoinduced increase in local polymerization. No such anomaly was observed with actin that had been labeled with fluorescein isothiocyanate (FITC). The FPR data are directly interpretable in terms of the fraction of labeled protein that is immobilized in the supramolecular assembly and in terms of the average diffusion coefficient of the mobile fraction. Our data are consistent with the "treadmill" model of actin polymerization, in that they show that actin is present under polymerizing conditions either as a high polymer or as monomer or low oligomer. We believe that the FPR technique can be applied to the study of many types of reconstituted motile or cytoskeletal systems in vitro or in vivo.

Actins↗
PubMed1981

Electrophoretic mobility distributions distinguish hairy cells from other mononuclear blood cells and provide evidence for the heterogeneity of normal monocytes.

The electrophoretic mobility distributions of hairy cells, normal monocytes. CLL, and normal lymphocytes isolated from blood were determined by electrophoretic light scattering. Values obtained for hairy cells, 1.52 X 10(-4) cm2/V . sec, were indistinguishable from that of normal monocytes. The mobility of CLL lymphocytes was similar to that of normal B cells. After exposure to neuraminidase, hairy cells revealed a homogeneous distribution with a reduced mobility of 0.55 X 10(-4) cm2/V . sec, while normal monocytes showed a heterogeneous distribution of electrophoretic mobilities suggestive of subpopulations. The electrokinetic behavior of hairy cells thus differs from that or normal and CLL lymphocytes before, and from that of monocytes after, treatment with neuraminidase. The hairy cell therefore possesses a distinct pattern of surface charge properties that clearly distinguish it from the circulating B cells, T cells, or monocytes.

B-Lymphocytes↗
PubMed1980

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.

Animals↗
PubMed1980

Electrokinetic properties of synaptic vesicles and synaptosomal membranes.

Using the technique of electrophoretic light scattering, we have measured the electrophoretic mobilities of synaptic vesicles and synaptosomal plasma membranes isolated from guinea-pig cerebral cortex. The electrophoretic mobility of synaptic vesicles is slightly greater than that of synaptosomal plasma membranes. Ca+2 and Mg+2 reduced the mobility of both species to the same extent at physiologically relevant concentrations (0-1 mM) and near-physiologic ionic strength. The extent of the reduction was not large (approximately 6% for synaptic vesicles in the presence of 100 mM KCl) at 1 mM divalent cation concentrations. At concentrations of approximately 2 mM and higher, Ca+2 reduced the mobility of synaptic vesicles more than did Mg/2. A similar but much smaller effect was observed in the case of synaptosomal plasma membranes. The addition of 1 mM Mg+2-ATP had no effect upon synaptic vesicle mobility either in the presence or absence of the ionophores nigericin or valinomycin. These data, together with earlier work (Siegel et al., 1978, Biophys. J. 22:341-346), demonstrate that substantial reduction of the average electrostatic surface charge density is not the most important role of divalent cations in promoting close approach of secretory granules and secretory cell membranes, and that it is certainly not the Ca+2-specific step in exocytosis.

Animals↗
PubMed1980

Adhesion of leukocytes to endothelium: roles of divalent cations, surface charge, chemotactic agents and substrate.

The attachment of polymorphonuclear leukocytes (PMN) to the endothelial lining of blood vessels is an important initial event in the acute inflammatory reaction. Experiments were carried out to examine some of the parameters of this adhesive interaction and the extent to which the substrate (endothelium) plays a role in controlling the process. Using a monolayer collection assay for adhesion, we found a requirement for divalent cations. Mn2+ produces maximal adhesion followed by Zn2+ greater than Ni2+ greater than Mg2+ greater than Ba2+ greater than Ca2+. Pretreatment of either the endothelium of PMN with chemotactic agents (zymosan-activated serum, a bacterial filtrate, C5a and formyl-methionyl-leucyl-phenylalanine) causes an increase in the attachment of PMN to endothelial monolayers. Modulations of net surface charge, as measured by laser Doppler electrophoresis, are not correlated to alterations in adhesion. Mn2+, which produces maximal adhesion, reduces the net surface charge to the same extent as Ca2+, which has the smallest effect on adhesion. Similarly, reductions in net surface charge are not responsible for the increases in adhesion due to the chemotactic agents because there are no differences in electrophoretic mobilities between cells treated with either chemotactic agents or their appropriate controls. Scatchard analysis of the binding of the chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, indicates the presence of high-affinity binding sites on the surface of the endothelium. The results suggest a role for the endothelium in mediating the adhesive interaction with the PMN.

Animals↗
PubMed1980

The role of negative charge in spontaneous aggregation of transformed and untransformed cell lines.

No correlation between net negative surface charge as determined by electrophoretic light-scattering techniques and the rates of spontaneous aggregation of 3T3MIT and SVPy 3T3MIT cells has been found. Neuraminidase treatment of both 3T3MIT cells and SVPy 3T3MIT cells causes a significant decrease in electrophoretic mobility but only the 3T3MIT cells show an increase in spontaneous aggregation. An increase in spontaneous aggregation of 3T3MIT cells is seen after growth in 200 mM urea for 18 h but no change in net surface charge occurs. The distribution of anionic sites on the membranes of cells was determined using the ultrastructural marker polycationized ferritin. The distribution of polycationized ferritin-binding sites was essentially identical for both cell lines under all conditions when they were labelled at 4 degrees C. When the cells were labelled with polycationized ferritin at 37 degrees C it was found that cells which have a high net rate of spontaneous aggregation also show rearrangement of anionic sites on their surface membrane. Clustering and rearrangement of anionic sites at 37 degrees C correlate with high rates of spontaneous aggregation.

Animals↗
PubMed1979

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggests that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.

Animals↗
PubMed1979

Macrophage response to concanavalin A: effect of surface crosslinking on the electrophoretic mobility distribution.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of concanavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea pig peritoneal macrophages. Con A, a tetrameric lectin, decreases slightly the mean mobility and increases by approximately 3-fold the width of the electrophoretic mobility distribution of resident macrophages. This effect can be abolished by alpha-methyl-D-mannoside, a hapten sugar of Con A. These effects were present in both low (0.010 M) and high (physiological, 0.15 M) ionic-strength media. Since lower ionic strengths correspond to a larger Debye screening distance, these data suggest that the alterations in the electrophoretic mobility distribution are not restricted to the outer portion of the glycocalyx. Succinyl-Con A, a dimeric derivative, was found to have no effect on the mobility distribution. However, the mean mobility decreased and the width increased over 4-fold when succinyl-Con A-treated macrophages were exposed to anti-Con A. These observations indicate that cross-linking of Con A receptors is an important process in the electrokinetic alterations of the macrophage surface. These results may have important consequences for the elucidation of the details of the endocytic mechanism.

Ascitic Fluid↗
PubMed1978

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.

Blood Protein Electrophoresis↗
PubMed1978

The effects of calcium2+ and magnesium2+ on the electrophoretic mobility of chromaffin granules measured by electrophoretic light scattering.

Electrophoretic light scattering was used to determine the electrophoretic mobility distributions of isolated bovine adrenal chromaffin granules as a function of divalent metal ion concentrations. Changes in the electrophoretic mobility reflected changes in the surface charge density of the granules. Ca2+ and Mg2+ (0.10--2.0 mM) were equally effective in reducing the electrophoretic mobilities. These findings are consistent with recent studies of the binding of Ca2+ and Mg2+ to the surface of chromaffin granules and are further evidence that the specific role of Ca2+ in exocytosis is due to effects other than the ability of Ca2+ to decrease the electrostatic repulsion between negatively charged membranes.

Adrenal Medulla↗
PubMed1978

Electrophoretic mobility distributions of normal human T and B lymphocytes and of peripheral blood lymphoblasts in acute lymphocytic leukemia: effects of neuraminidase and of solvent ionic strength.

Human T and B lymphocytes were found to be distinguishable on the basis of electrophoretic mobility, with the T cells having the higher mobility, in agreement with previous reports. The effects of the enzyme neuraminidase on the electrophoretic mobilities of T and B lymphocytes were determined. T lymphocytes showed a greater decrease in electrophoretic mobility after neuraminidase treatment; the relative mobilities of T and B cells were reversed by neuraminidase treatment, and the electrophoretic distinguishability was enhanced. The electrophoretic mobility distributions of lymphoblasts from patients with acute lymphocytic leukemia were found to differ from those of normal cells in their response to neuraminidase treatment and to changes in solution ionic strength. These results imply that the surface structure of the leukemic cells differs from that of either T or B lymphocytes from normal donors.

B-Lymphocytes↗
PubMed1977

Velocity distributions of the streaming protoplasm in Nitella flexilis.

Laser light is Doppler-shifted in frequency by the streaming endoplasm of living cells of Nitella flexilis. The frequency spectrum of the scattered light can be interpreted as the histogram of velocities within the organism, with the exception of the intense low-frequency portion of the spectrum. We demonstrate that the lowest-frequency component is the result of amplitude modulation of the scattered light by the array of chloroplasts in the cell. Measurement of the streaming endoplasm in a photobleached "window" region allows correction of the frequency distribution for the modulation component. The complete velocity histogram for the streaming endoplasm is calculated directly from the corrected frequency distribution. Measurements of vacuolar and endoplasmic motions show that the tonoplast, the membrane separating the vacuole and the endoplasm, seems to be flowing along with the endoplasm and vacuolar sap. Placing the cell in medium containing ATP in concentrations greater than 10(-3) M greatly increases the contribution of low velocities to the velocity histogram. Cytochalasin B at high dosages (10-50 mug/ml) does not noticably change the shape of the velocity histogram, while at low dosages (1 mug/ml) there is an increase in the contribution of low velocities to the velocity histogram. Colchicine in high concentrations (1%) has no observable effect on the velocity histogram.

Adenosine Triphosphate↗