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Biomedical subjects

D Axelrod

Publications and source records attributed to D Axelrod.

At least 73 records · Page 4Linked to original sources
PubMed1983

Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes.

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AcChoR) are found to be aggregated and immobilized in endogenous clusters while the remainder are diffusely distributed and partially mobile. This paper reports that AcChoR in clusters can be gathered from AcChoR in diffuse areas during the course of normal myotube development. AcChoR were fluorescently labeled with rhodamine-conjugated alpha-bungarotoxin, and all existing clusters in a circumscribed region of the culture dish were irreversibly photobleached by a slightly defocused laser beam, the movement of which was controlled by a lens mounted on a joystick translator. This procedure leaves intact only the fluorescent label on the diffusely distributed AcChoR. Observation of the myotubes after several hours of incubation revealed cluster fluorescence redevelopment. This cluster fluorescence must have consisted of AcChoR that previously were diffusely distributed. The majority (but not all) of cluster fluorescence redevelopment occurred in the location of a previously bleached cluster. About half of the redeveloped clusters have an annular shape. The major conclusions of this study are (i) diffusely distributed AcChoR can become clustered; (ii) endogenous clusters appear to form, at least in part, by "trapping" receptors as they diffuse in from surrounding regions; (iii) cluster formation is an ongoing process in cultured rat myotubes; and (iv) colchicine (a microtubule-disrupting agent) inhibits cluster formation.

Animals↗
PubMed1983

Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation.

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AChR) are found aggregated and immobilized in endogenous clusters. The remaining receptors are diffusely distributed over the cell membrane and the majority of these are free to diffuse in the plane of the membrane. This study correlates the mobility of AChR (as measured with the fluorescence photobleaching recovery technique, FPR) with the detergent extractability of this receptor. Gentle detergent extraction of the cells removes the lipid membrane and the soluble cytoplasmic proteins but leaves an intact cytoskeletal framework on the substrate. Two studies indicate a correlation between mobility and extractability: (a) mobility of diffusely distributed AChR decreases as myotubes age in culture; previous work showed that extractability of AChR decreases as myotubes age in culture (Prives, J., C. Christian, S. Penman, and K. Olden, 1980, In Tissue Culture in Neurobiology, E. Giacobini, A. Vernadakis, and A. Shahar, editors, Raven Press, New York, 35-52); (b) mobility of clustered AChR increases when cells are treated with metabolic inhibitors such as sodium azide (NaN3); extractability of clustered AChR also increases with this treatment. From these results we suggest the involvement of a cytoskeletal framework in the immobilization of AChR on the cell surface.

Animals↗
PubMed1983

Total internal reflection fluorescent microscopy.

This review discusses applications of fluorescence microscopy using totally internally reflected excitation light. When totally internally reflected in a transparent solid at its interface liquid, the excitation light beam penetrates only a short distance into the liquid. This surface electromagnetic field, called the 'evanescent wave', can selectively excite fluorescent molecules in the liquid near the interface. Total internal reflection fluorescence (TIRF) has been used to examine the cell/substrate contact regions of primary cultured rat myotubes with acetylcholine receptors labelled by fluorescent alpha-bungarotoxin and human skin fibroblasts labelled with a membrane-incorporated fluorescent lipid. TIRF examination of cell/surface contacts dramatically reduces background from cell autofluorescence and debris. TIRF has also been combined with fluorescence photobleaching recovery and correlation spectroscopy to measure the chemical kinetic binding rates and surface diffusion constant of fluorescent labelled serum protein binding (at equilibrium) to a surface.

Animals↗
PubMed1981

Studies on the localization of newly added membrane in growing neurites.

Explant culture of the adult goldfish retina results in vigorous neuritic growth, provided that the optic nerve has been previously crushed in vivo. The site of new membrane addition in these growing neurites was studied with a a membrane-binding lectin Concanavalin A (Con A) in conjunction with a fluorescent indirect antibody method. Explant cultures were labeled with Con A followed by rabbit antibodies to Con A. This treatment was shown to immobilize Con A receptors while permitting growth. Twenty-four hours later, the washed preparation was treated with fluorescent goat antirabbit antibodies. The bound fluorescent antibodies appeared as a pattern of contiguous fluorescent speckles confined to the area of old growth while regions near the distal end of the neurite were not fluorescent. These results are compatible with the hypothesis that new membrane is added in the region of the growing tip of the neurite.

Animals↗
PubMed1981

Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics.

A simple, zero-cost, reversible modification of a bright field microscope permits visualization of phase gradients in cells by transmitted illumination, yielding a Nomarski-like effect. This modification, based on schlieren optics, is simultaneously compatible with high-aperture epi-illumination fluorescence excitation. For many objectives that are intended for use in fluorescence work, but are unavailable in phase contrast versions, the modification provides a simple means for locating cells in culture with good image contrast and resolution.

Animals↗
PubMed1981

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy.

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).

Kinetics↗
PubMed1981

Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics.

The total internal reflection/fluorescence photobleaching recovery (TIR/FPR) technique (Thompson et al. 1981. Biophys. J. 33:435) is used to study adsorbed bovine serum albumin dynamics at a quartz glass/aqueous buffer interface. Adsorbed fluorescent labeled protein is bleached by a brief flash of the evanescent wave of a focused totally internally reflected laser beam. The rates of adsorption/desorption and surface diffusion determine the subsequent fluorescence recovery. The protein surface concentration is low enough to be proportional to the observed fluorescence and high enough to insure that the observed recovery rates arise mainly from adsorbed rather than bulk protein dynamics. The photobleaching recovery curves for rhodamine-labeled bovine serum albumin reveal both an irreversibly bound state and a multiplicity of reversibly bound states. The relative amount of reversible to irreversible adsorption increases with increasing bulk protein concentration. Since the adsorbed protein concentration appears to be too high to pack into a homogeneous surface monolayer, the wide range of desorption rates possibly results from multiple layers of protein on the surface. Comparison of the fluorescence recovery curves obtained with various focused laser beam widths suggests that some of the reversibly bound bovine serum albumin molecules can surface diffuse. Aside from their relevance to the surface chemistry of blood, these results demonstrate the feasibility of the TIR/FPR technique for measuring molecular dynamics on solid surfaces.

Adsorption↗
PubMed1981

A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells.

A factor or factors released by cultured NG108-15 neuroblastoma X glioma hybrid cells and added to the medium of rat myotube primary cultures was found to immobilize some of the previously mobile acetylcholine receptors in the myotube membrane. Partial receptor immobilization occurred within 3 h after the beginning of treatment with the NG108-15-conditioned medium factor and persisted for at least 24 h of continuous treatment. A similarly derived conditioned medium concentrate from the non-neuronal parent glioma cell line did not immobilize receptors, relative to untreated controls. Acetylcholine receptors were visualized by fluorescent alpha-bungarotoxin and their lateral motion was observed by the technique of fluorescence photobleaching recovery.

Animals↗
PubMed1981

Cell-substrate contacts illuminated by total internal reflection fluorescence.

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.

Animals↗
PubMed1980

Histological localization of binding sites of alpha-bungarotoxin and of antibodies specific to acetylcholine receptor in goldfish optic nerve and tectum.

Goldfish optic nerve as well as ganglion cell neurites grown in culture selectively bind rhodamine-labeled alpha-bungarotoxin following tissue fixation. Binding is competed for by unlabeled bungarotoxin, by carbamylcholine and tubocurarine, but not by atropine. In cross-sections, the label is seen confined to axonal bundles. The binding is not detectable without prior fixation and is very faint in brain sections, even after fixation. To further establish the nature of the binding, immunocytochemical studies were performed, taking advantage of a high cross-reactivity found between goldfish brain and antibodies against eel acetylcholine receptor (AChR). Antigenic sites were detected by an indirect unlabeled antibody complexed to horseradish peroxidase. Anti-AChR antibody binding to optic nerve and neurites in culture correlated with that seen with alpha-bungarotoxin. Binding of anti-AChR was observed in the brain, and was reduced in the denervated tectum following unilateral optic nerve crush or enucleation. The results are discussed in relation to functions of receptor proteins in the retinotectal system.

Acetylcholine↗
PubMed1980

Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane.

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperature (-5 to 5 degrees C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40 degrees C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers. Whole erythrocytes were depleted of 30--50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3'-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot. The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.

Carbocyanines↗
PubMed1980

Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.

A biotinylated derivative of alpha-bungarotoxin and tetramethylrhodamine-labeled avidin were employed to fluorescence label the acetylcholine receptors (AcChoR) on the surface of rat myotubes in primary culture. Because of the multivalency of both the biotinylated bungarotoxin and the avidin, this treatment extensivey crosslinks the AcChoR. AcChoR crosslinking immobilizes more than 90% of the normally laterally mobile AcChoR as verified by the fluorescence photobleaching recovery technique; it also redistributes the AcChoR into visible micropatches. Biotinylated alpha-bungarotoxin/avidin-induced AcChoR crosslinking greatly accelerates the rate of internalization of surface AcChoR; this rapid internalization affects both the normally immobile AcChoR in areas of diffuse distribution and the normally immobile AcChoR in preexisting patches. The peculiar pattern of fluorescent avidin binding to AcChoR patches previously bound with biotinylated bungarotoxin suggests that almost all AcChoR patches are in very close contact (< 70 A) with the glass substrate. AcChoR immobilization leads to a partial immobilization of concanavalin A receptors in the myotube membrane.

Animals↗
PubMed1979

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

Carbocyanines↗
PubMed1978

Activities of types A and B MAO and catechol-o-methyltransferase in blood cells and skin fibroblasts of normal and chronic schizophrenic subjects.

We assayed activities of monoamine oxidase (MAO) type B in blood platelets and type A (and B) in fibroblasts cultured from punch biopsy specimens of skin, as well as of catechol-O-methyltransferase (COMT) in erythrocytes and fibroblasts. Fibroblasts contained moderate amounts of both forms of MAO (types A and B) found in human brain and large amounts of COMT activity. Activities of both enzymes correlated poorly between fibroblasts and blood cells. Comparing carefully diagnosed chronic schizophrenics with age-matched normal young men, we found no difference in these biochemical variables, nor could we distinguish patients with paranoid symptoms. In contrast, we confirmed markedly lower MAO activities in platelet samples from chronic patients provided by colleagues at the National Institute of Mental Health. Results concerning MAO and COMT activities are now sufficiently inconsistently characteristic of schizophrenics as to question their clinical applicability and to indicate a need for further critical evaluation, with special attention to diagnosis, matching of subjects, and effects of possible spurious environmental variables.

Adolescent↗