Biomedical subjects

W W Webb

Publications and source records attributed to W W Webb.

At least 19 recordsLinked to original sources

PubMed1992

Forward and reverse transduction at the limit of sensitivity studied by correlating electrical and mechanical fluctuations in frog saccular hair cells.

The spontaneous fluctuations of the intracellular voltage and the position of the sensory hairbundle were measured concurrently using intracellular microelectrodes and an optical differential micro interferometer. Magnitude and frequency distribution of the hair bundles' spontaneous motion suggest that it consists mostly of Brownian motion. The electrical noise, however, exceeds the value expected for thermal Johnson noise by several orders of magnitude, and its frequency distribution reflects the transduction tuning properties of the hair cells. Frequently, a strong correlation was observed between the fluctuations of the hair bundle position and the intracellular electrical noise. From the properties of the correlation and from experiments involving mechanical stimulation we conclude that in most cases mechano-electrical transduction of the bundles' Brownian motion causes this correlation. Small signal transduction sensitivities ranged from 18 to 500 microV/nm. Bundle motion that was observed in response to current injection in more than half of the cells suggests the existence of a fast reverse (electro-mechanical) transduction mechanism to be common in these cells. The sensitivities could be as high as 600 pm of bundle deflection per millivolt of membrane potential change. In a significant minority (4 in 44) of cells, all showing excess electrical noise, we found 'non-causal' components of the electro-mechanical correlation, and in two of those cells narrow-band bundle motion in excess of their thermal motion at frequencies coincident with peaks in the intracellular noise was observed.

Animals↗

PubMed1992

Effects of protein concentration on IgE receptor mobility in rat basophilic leukemia cell plasma membranes.

The ability of variations of membrane protein concentrations to modulate the lateral diffusion rate of an exemplary membrane protein has been studied in healthy and osmotically shocked cultured cells of the rat basophilic leukemia cell line, 2H3 subclone. Cell surface protein was redistributed by the method of in situ electrophoresis; exposure to electric fields of 1.25-5 V/cm results in cathodal migration of the majority of the surface proteins on this cell type (Ryan, T. A., J. Myers, D. Holowka, B. Baird, and W. W. Webb. Science [Wash. DC]. 239:61-64). Even in these small fields, the steady-state distribution becomes "crowded" with more than an 80% protein occupancy of accessible membrane area at the cathodal end of these spheroidal cells, and the anodal end becomes significantly depleted. We have employed fringe pattern fluorescence photobleaching with CCD imaging detection to measure lateral diffusion coefficients of the liganded IgE receptor on both crowded and uncrowded regions of individual rat basophilic leukemia cells. We find no significant difference in lateral diffusion rates in these regions. Cells swollen by hypoosmotic stress exhibit faster diffusion overall, with the uncrowded regions having a significantly greater increase in diffusion coefficient than the crowded regions. These results are consistent with the partial or total release of cytoskeletal constraints to membrane protein diffusion induced by osmotic stress.

Animals↗

PubMed1992

Mechanical response of frog saccular hair bundles to the aminoglycoside block of mechanoelectrical transduction.

1. Deflections of the mechanosensory hair bundles on frog saccular hair cells were measured interferometrically, with submillisecond temporal and submicrometer spatial resolution, and with subnanometer displacement sensitivity. 2. The direction of the initial bundle deflection (toward the taller stereocilia) in response to a sudden application of aminoglycoside antibiotics shows that the mechanosensory channels are blocked in their mechanically open state. 3. The magnitude of the initial deflection is consistent with published data on the gating swing as derived from the gating compliance. 4. A delayed relaxation and frequently a reversal of the initial deflection were observed and are attributed to the previously reported mechanical adaptation mechanism, which is at least partially controlled by the influx of Ca2+ through the transduction channels. 5. Increases of low-frequency spontaneous motion were found at intermediate blocker concentrations. They can be well accounted for by the fluctuating force exerted on the bundle by the random binding and unbinding of blocker molecules. 6. The mechanical response of the hair bundle to aminoglycosides may be related to their acute and specific ototoxicity.

Aminoglycosides↗

PubMed1991

Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy.

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-ceramide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PK1 porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 micron). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PK1 cells, about 2.5% in 3T3 cells and L5 cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1990

Two-photon laser scanning fluorescence microscopy.

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

Animals↗

PubMed1990

Heterogeneity of personality traits in massive obesity and outcome prediction of bariatric surgery.

The search for heterogeneity of personality characteristics amongst the massively obese is extended to a larger sample (n = 215) of both males (n = 83) and females (n = 132). The Minnesota Multiphasic Personality Inventory standard scales serve as the data base for a cluster analysis which has yielded four clusters: cluster 1 (n = 36) where all scales fell well within normal limits; cluster 2 (n = 112) was coded as 3-1 and was also within normal limits; cluster 3 (n = 44) was coded as spike 4; and cluster 4 (n = 23) was coded as 2-1-7-3-8-4. Cluster membership was not related to surgeons' four-year follow-up overall outcome rating. Discriminant function analysis yielded two variables (patient age and MMPI scale 1 T-score) which were significant predictors of subsequent outcome. The results suggest that there is heterogeneity of personality traits among the massively obese and offer encouragement to the search for variables that are even more effective predictors of the outcome of bariatric surgery.

Cluster Analysis↗

PubMed1989

Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells.

Fura-2 fluorescence in single rat basophilic leukemia cells was monitored to study the rise in intracellular free ionized calcium ([Ca2+]i) produced by aggregation of immunoglobulin E receptors. Repetitive transient increases in [Ca2+]i were induced by antigen stimulation and were measured using digital video imaging microscopy at high time resolution. The [Ca2+]i oscillations were not dependent upon changes in the membrane potential of the cells and were observed in cells stimulated with antigen either with or without extracellular Ca2+. Transient oscillations in [Ca2+]i were also observed when calcium influx was blocked with La3+. These results suggested that during antigen stimulation of cells under normal physiological conditions, release of Ca2+ from intracellular stores makes an important contribution to the initial increase in [Ca2+]i. Oscillations in [Ca2+]i are not induced by elevating [Ca2+]i with the calcium ionophore ionomycin. Mitochondrial calcium buffering is not required for [Ca2+]i oscillations to occur. The results show that rat basophilic leukemia cells have significant stores of calcium and that release of calcium from these stores can participate in both the initial rise and the oscillations in [Ca2+]i.

Adenosine Triphosphate↗

PubMed1989

Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand.

A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.

Carbachol↗

PubMed1989

Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer.

By optically probing with a focused, low-power laser beam, we measured the spontaneous deflection fluctuations of the sensory hair bundles on frog saccular hair cells with a sensitivity of about 1 pm/square root of Hz. The preparation was illuminated by two orthogonally polarized laser beams separated by only about 0.2 microns at their foci in the structure under investigation. Slight movement of the object from one beam toward the other caused a change of the phase difference between the transmitted beams and an intensity modulation at the detector where the beams interfered. Maintenance of the health of the cells and function of the transduction mechanism were occasionally confirmed by measuring the intracellular resting potential and the sensitivity of transduction. The root-mean-square (rms) displacement of approximately 3.5 nm at a hair bundle's tip suggests a stiffness of about 350 microN/m, in agreement with measurements made with a probe attached to a bundle's tip. The spectra resemble those of overdamped harmonic oscillators with roll-off frequencies between 200 and 800 Hz. Because the roll-off frequencies depended strongly on the viscosity of the bathing medium, we conclude that hair-bundle motion is mainly damped by the surrounding fluid.

Animals↗

PubMed1988

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: studies on individual cells using quantitative digital video fluorescence microscopy.

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.

Adenosine Triphosphate↗

PubMed1988

Molecular crowding on the cell surface.

Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions of proteins in applied potential gradients. The fraction of accessible surface occupied by mobile surface proteins can be accurately represented by including steric exclusion in the statistical thermodynamic analysis of the data. The analyses revealed enhanced, concentration-dependent activity coefficients, implying unanticipated thermodynamic activity even at typical cell surface receptor concentrations.

Animals↗

PubMed1988

Imaging asynchronous changes in intracellular Ca2+ in individual stimulated tumor mast cells.

Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of mediators of immediate hypersensitivity by rat basophilic leukemia (RBL) cells, a mast cell line. Digital fluorescence ratio imaging of fura-2 was used to measure [Ca2+]i in individual RBL cells. Changes in [Ca2+]i that occurred in response to crosslinking of IgE receptors on the cell surface or to application of the Ca2+ ionophore ionomycin were studied. Stimulation of RBL cells with antigen resulted in rapid increases in [Ca2+]i following lag times that varied widely from cell to cell. Simple averaging of the Ca2+ responses of many cells yielded a gradual response profile that closely resembled that of suspensions of cells measured in the fluorometer. The results show that single cells can respond much more rapidly to antigen than has previously been suggested by studies on populations of cells. The lag time between addition of antigen and initiation of the increase in [Ca2+]i varied considerably between cells in the same field of view. Both the rise time and the variability and average duration of the lag time increased with decreasing antigen concentration.

Animals↗

PubMed1987

Fluorescence light microscopy of F-actin in retinal rods and glial cells.

The actin cytoskeleton of rod photoreceptors and glial cells in toad retina has been directly viewed using fluorescence microscopy of cells labeled with a potent phallotoxin that specifically binds to F-actin. The three-dimensional organization of this cytoskeletal protein consists of actin filaments, which course through the inner segment and end at the tips of the calycal processes surrounding the base of the outer segment. A transverse layer of actin staining is also observed at the base of rod outer segments in the region where new discs are formed. At the level of the external limiting membrane, evidence has been found for rings of actin within the glial cells that surround the photoreceptors. These actin rings form a structural meshwork in which photoreceptor cells are embedded.

Actins↗

PubMed1986

Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells.

We have recently shown that small oligomers of IgE bound to univalent receptors for IgE on the surface of rat basophilic leukemia cells induce extensive aggregation of the receptors at 4 degrees C into patches resolvable by fluorescence microscopy and that this does not occur with monomeric IgE (Menon, A. K., D. Holowka, and B. Baird, 1984, J. Cell Biol. 98:577-583). Here we use fluorescence photobleaching recovery measurements to show that receptor oligomerization by this means is accompanied by a dramatic reduction of receptor lateral mobility, and that this immobilization occurs even when the clustering is not microscopically detectable. Furthermore, the degree of immobility induced by a particular oligomer fraction from a gel filtration column correlates positively with its ability to trigger cellular degranulation, whereas receptors labeled with monomeric IgE have no triggering activity and exhibit typical membrane protein mobility. The slow, large-scale oligomer-induced clustering appears to be a long term consequence of earlier selective interactions that result in receptor immobilization, and this highly clustered state provides a competent, noninhibitory triggering signal resulting in cellular degranulation upon warming to 37 degrees C. We conclude that even limited clustering of IgE receptors on rat basophilic leukemia cells induces interactions with other cellular components that constrain receptor mobility and eventually cause massive coalescence of the clusters. These primary selective interactions occurring at the level of receptor oligomers or small clusters of oligomers that result in immobilization may play a role in triggering cellular degranulation.

Animals↗

PubMed1986

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization.

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.

Animals↗

PubMed1986

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin.

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.

Actinin↗

PubMed1985

Coupling of ATP synthesis to reversal of rat liver microsomal Ca2+-ATPase.

The reversal of the rat liver microsomal Ca2+-ATPase transport cycle was studied. Microsomes were loaded with 45Ca2+ (approximately 30 nmol/mg of protein) in an ATP-dependent process, and the time dependency of the microsomal 45Ca2+ efflux was determined with various ADP and inorganic phosphate (Pi) concentrations. Pseudo-first-order rate constants (K'e) for 45Ca2+ efflux were determined. Although there was considerable 45Ca2+ efflux in the absence of added ADP or Pi, the addition of ADP or Pi alone had minimal effects upon the K'e; in contrast, a 2.5-fold increase in the K'e was observed in the presence of both ADP and Pi. The apparent Km values for ADP and Pi were 4 microM and 0.22 mM, respectively. Stimulation of 45Ca2+ efflux by ADP and Pi was associated with ATP synthesis. The calcium ionophore A23187 prevented ATP synthesis, which indicates that the Ca2+ gradient facilitates the coupling of ATP synthesis to Ca2+ efflux.

Adenosine Diphosphate↗

PubMed1985

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts.

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.

Cell Membrane↗