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Results for “DILS”

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At least 127 records · Page 7Linked to original sources

PubMed1998

Inhibition of nuclear factor kappaB activation attenuates apoptosis resistance in lymphoid cells.

Death-inducing ligands (DILs) such as tumor necrosis factor alpha (TNFalpha) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor kappaB (NFkappaB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFkappaB in lymphoid cell lines in a kinetic similar to TNFalpha. NFkappaB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFkappaB activity on apoptosis mediated by TRAIL, CD95, TNFalpha, or doxorubicin, NFkappaB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IkappaBalpha. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFkappaB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFkappaB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment.

Antibiotics, Antineoplastic↗

PubMed1994

Antibodies to HMG proteins in patients with drug-induced autoimmunity.

OBJECTIVE: To determine the prevalence of autoantibodies to high-mobility group (HMG) proteins in sera from patients with drug-induced lupus (DIL). METHODS: Forty-two patients who developed autoantibodies and/or lupus after treatment with procainamide or other drugs were tested for HMG autoantibodies by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Twenty-eight of the 42 sera (67%) bound HMG-14 and/or HMG-17. In comparison, 9 of 42 (21%) bound HMG-1 and/or HMG-2. There was a good correlation between ELISA results and binding on immunoblots. CONCLUSION: The high prevalence of antibodies to the nucleosomal core HMGs (HMG-14 and HMG-17) in DIL patients adds evidence implicating nucleosomes as immunogens in drug-induced autoimmunity.

Acebutolol↗

PubMed1995

Preservation of topography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats.

In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV-VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex.

Afferent Pathways↗

PubMed1991

Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel.

Interleukin-1 receptor antagonist (IL-1ra), a true antagonist of the interleukin-1 (IL-1) receptors, is released by activated macrophages and binds specifically to the IL-1 receptors without triggering IL-1 effects. Following peripheral nerve axotomy, activated macrophages release IL-1, which induces the expression of nerve growth factor (NGF) mRNA in Schwann cells. IL-1ra may therefore impede peripheral nerve regeneration by blocking the NGF-mediated effect of IL-1. Peripheral nerve regeneration occurring through polymeric guidance channels releasing IL-1ra was investigated in a 4-mm gap transected mouse sciatic nerve model. Cohorts of five animals were implanted with tubes releasing either bovine serum albumin (BSA), BSA with IL-1ra, or BSA with deactivated IL-1ra (dIL-1ra) for 4 weeks. In vitro release kinetics indicated that after an initial burst, IL-1ra release was linear for the next 3 1/2 weeks. Following implantation of a polymeric guidance channel, a regenerated cable bridged the nerve gap in all animals. The cables were similar in size and were composed of nerve microfascicles containing both unmyelinated and myelinated axons in association with their Schwann cells. Tissue regenerated in tubes releasing BSA-IL-1ra contained, however, significantly fewer myelinated and unmyelinated axons and blood vessels than did tubes releasing BSA alone or BSA-dIL-1ra. We conclude that a naturally occurring antagonist of IL-1 receptors impedes peripheral nerve regeneration, suggesting that macrophages play an essential role in controlling peripheral nerve regeneration through the release of stimulatory and/or inhibitory molecules.

Animals↗

PubMed1995

Safety, tolerability, and pharmacokinetic actions of diltiazem in pediatric liver transplant recipients on cyclosporine.

Thirty-two children who had undergone liver transplantation were paired according to their posttransplantation duration, renal function, and diagnoses when possible and randomized either to continue nifedipine (NIF group) or switch to diltiazem (DIL group), in addition to continuing their usual immunosuppressive medications. The cases were followed prospectively regarding diltiazem tolerance, cyclosporine dose requirements, effect on cyclosporine kinetics, diltiazem kinetics, as well as effect on renal function. Diltiazem was well tolerated at a dose of 3 mg to 6.4 mg/kg/day (max 180 mg/day) with infrequent self-limited mild side effects. Cyclosporine daily dose was reduced by a mean of 36.7% and 38.3% at 3 and 6 months, respectively, in the DIL group to achieve target trough cyclosporine levels without modifying liver function. No significant difference in renal function was observed after 3 to 6 months in either group based on blood urea nitrogen and creatinine levels and glomerular filtration rate by the DTPA method. Diltiazem appears to be well tolerated in children and allows for substantial dose reductions of CSA without apparent effects on liver graft function.

Adolescent↗

PubMed1990

Effects of calcium channel blockers on the development of early rat postimplantation embryos in culture.

Rat embryos (9.5-day-old) were cultured for 48 h in the presence of nifedipine (NIF), nimodipine (NIM), nitrendipine (NIT), gallopamil HCl (GAL), verapamil HCl (VER) and diltiazem HCl (DIL). The effects on growth and morphogenetic differentiation in vitro were monitored. Dose-response relationships were evaluated, including an assessment of the "no-observed-effect-level" (NOEL) or the "lowest-observed-effect-level" (LOEL), and the lowest concentration tested inducing abnormalities in 100% of the embryos ("100% EL"). The morphological alterations observed at the highest concentrations were very similar for all six drugs. The abnormalities concerned yolk sac circulation and morphology, as well as heartbeat, the morphology of the heart, head, neural tube, or forelimbs, and the shape of the embryo. The abnormal embryos were also growth retarded (decrease in protein content and crown-rump length). Interference with calcium channel functions seems to represent an interesting model for studying a special kind of abnormal prenatal development, especially the differentiation of certain mesenchymal structures. The concentration ranges between NOELs and 100% ELs were found to be: NIM = 0.1-1 microgram/ml; NIT and VER = 1-10 micrograms/ml; DIL = 1-30 micrograms/ml, and LOELs-100% ELs were: GAL = 1-10 micrograms/ml; NIF = 10-30 micrograms/ml.

Abnormalities, Drug-Induced↗

PubMed1996

Ultrasound-assisted synthesis of santalbic acid and a study of triacylglycerol species in Santalum album (Linn.) seed oil.

Methyl ricinoleate (1) was treated with bromine and the dibromo derivative (2) was reacted with ethanolic KOH under ultrasonic irradiation to give 12-hydroxy-octadec-9-ynoic acid upon acidification with dil. HCI. The latter compound was methylated with BF3/methanol to give methyl 12-hydroxy-octadec-9-ynoate (3). Compound 3 was treated with methanesulfonyl chloride in the presence of triethylamine in CH2Cl2 to give methyl 12-mesyloxy-octadec-9-ynoate (4). Reaction of methyl 12-mesyloxy-octadec-9-ynoate with aqueous KOH under ultrasonic irradiation (20 kHz) gave (11E)-octadecen-9-ynoic acid (5, santalbic acid, 40%) and (11Z)-octadecen-9-ynoic acid (6, 60%) on acidification with dil. HCI. These isomers were separated by urea fractionation. The 13C nuclear magnetic resonance (NMR) spectroscopic properties of the methyl ester and the triacylglycerol (TAG) esters of these enynoic fatty acid isomers were studied. The carbon shifts of the unsaturated carbon nuclei of the methyl ester of the E-isomer were unambiguously assigned as 88.547 (C-9), 79.287 (C-10), 109.760 (C-11), and 143.450 (C-12) ppm, while the unsaturated carbon shifts of the (Z)-enynoate isomer appeared at 94.277 (C-9), 77.561 (C-10), 109.297 (C-11), and 142.668 (C-12) ppm. In the 13C NMR spectral analysis of the TAG molecules of type AAA containing either the (Z)- or (E)-enyne fatty acid, the C-1 to C-6 carbon atoms on the alpha- and beta-acyl positions were differentiated. The unsaturated carbon atoms in the alpha- and beta-acyl chains were also resolved into two signals except that of the C-11 olefinic carbon. Sandal (Santalum album) wood seed oil (a source of santalbic acid) was separated by silica chromatography into three fractions. The least polar fraction (7.2 wt%) contained TAG which had a random distribution of saturated and unsaturated fatty acids, of which oleic acid (69%) was the predominant component. The second fraction (3.8 wt%) contained santalbic acid (58%) and oleic acid (28%) together with some other normal fatty acids. Santalbic acid in this fraction was found in both the alpha- and beta-acyl positions of the glycerol "backbone." The most polar fraction (89 wt%) consisted of TAG containing santalbic acid only. The distribution of the various fatty acids on the glycerol "backbone" was supported by the results from the 13C NMR spectroscopic analysis.

Carbon Isotopes↗

PubMed1993

Epitope mapping of histone 5 (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera.

To define the linear epitopes on H5 that react with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) sera, concurrent overlapping hexameric peptides corresponding to the sequence of H5 were synthesized by stepwise elongation of the polypeptide chains on polyethylene supports. The hexapeptides were tested for reactivity with 8 SLE and 8 DIL sera using an enzyme linked immunosorbent assay (ELISA). SLE and hydralazine-induced lupus (HIL) antibodies were most reactive with peptide 45 (SSRQSI) and patients with procainamide-induced lupus (PIL) were most reactive with peptide 24 (SHPTYS). The epitopes of highest reactivity were in the globular domain of H5. Low reactivity was observed with carboxyl terminal peptides. These findings differ from immunoblotting studies of protease cleaved peptides which have previously shown that the H5 determinants are in the carboxyl terminus.

Amino Acid Sequence↗

PubMed1994

A population of myogenic cells derived from the mouse neural tube.

Embryonic mouse neural tubes produce a variety of terminally differentiated cells in vitro, mostly neurons and glia. We report here that some of these cells differentiate into skeletal muscle cells. The possibility of mesoderm contamination was ruled out as follows. First, Dil+ muscle cells were present in cultures from a Dil-labeled neuroepithelium. Second, a small fraction of cultured neural tube cells coexpressed muscle myosin and neuronal beta III tubulin within the same cell. Third, embryos generated from embryonic stem cells in which nlacZ was targeted into the myogenic gene myf-5 expressed nlacZ in a localized region of the neural tube. These myf-5+ cells coexpress neuronal and muscle markers in culture. The developmental significance of this phenomenon is discussed in the context of overlapping regulatory networks between myogenesis and neurogenesis.

Animals↗

PubMed1992

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.

Animals↗

PubMed1998

Non-adrenergic, non-cholinergic neurons innervating the guinea-pig trachea are located in the oesophagus: evidence from retrograde neuronal tracing.

The possibility that non-adrenergic, non-cholinergic (NANC) neurons innervating the guinea-pig trachea may be located within the oesophagus has been investigated using an in vitro retrograde tracing technique. The cervical trachea and oesophagus were excised from guinea-pigs and Dil was applied to a 5 mm region of the trachealis muscle. These preparations were maintained in organotypic culture for 3 days and processed for immunohistochemistry. A mean of 44 (4 neural cell bodies in the oesophageal myenteric plexus were found to be labelled by Dil. The vast majority of these neurons contained nitric oxide synthase, vasoactive intestinal polypeptide and neuropeptide Y. It is suggested that the population of neurons identified in this study are postganglionic parasympathetic neurons mediating NANC relaxation of the trachealis muscle in this species.

Animals↗

PubMed1998

Synthesis and biological activity of chimeric structures derived from the cytotoxic natural compounds dolastatin 10 and dolastatin 15.

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were synthesized in order to evaluate the possible equivalence of these units. These compounds can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the former and the C-terminal part of the latter and vice versa. Both analogues exhibited a marked decrease in their cytotoxic activity but showed similar differential cytotoxicity with regard to the cell lines assayed compared with the parent compounds. HT-29 cell line was the least sensitive one. However, this activity was in the nanomolar level and close to that of vincristine. The differences in their effect on tubulin polymerization were less pronounced. We confirmed the already known crucial role of the Dil residue in this assay. The nonequivalence of the Dil unit and the MeVal-Pro dipeptide probably reflects modification in the relative positions of the N-dimethylamino and the phenyl moieties.

Animals↗

PubMed1992

Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity.

The nature and physiological importance of high-density lipoprotein (HDL) binding sites on unstimulated (resting) and mitogen-activated (blast) human peripheral blood lymphocytes were investigated. Specific HDL binding on resting and blast T-lymphocytes was saturable at 50 micrograms of 125I-HDL/ml and of high affinity, with Kd values of 8.1 x 10(-8) M and 6.5 x 10(-8) M, respectively, and Bmax. values of 79 ng and 180 ng/mg of cell protein respectively at 4 degrees C. Binding of HDL double-labelled with fluorescent dioctadecylindocarbocyanine (Dil) and isotope (125I) as well as of single fluorescence- or isotope-labelled HDL was inhibited competitively by HDL apoproteins. Studies of the cholesterol flux between the cells and HDL showed that HDL, low-density lipoprotein (LDL) or BSA at a concentration of 100 micrograms/ml in the tissue culture medium did not result in a significant difference in exogenous [3H]cholesterol efflux from the cell membrane at 37 degrees C. Proliferating T-blasts incorporated more cholesterol from HDL or LDL than did resting lymphocytes. When the cells were pulsed with 125I-HDL and chased in fresh lipid-free medium, up to 80% of the radioactivity released was not precipitable with trichloroacetic acid. This percentage decreased in a competitive manner when unlabelled HDL was present in the chase incubation medium. Finally, cultivation of lymphocytes with conditioned medium from macrophages increased Dil-HDL binding/uptake, while it was decreased by mevinolin-induced inhibition of hydroxymethylglutaryl-coA reductase. In conclusion, human lymphocytes possess a HDL binding site (receptor) responsible for lipid binding/uptake and concomitant internalization and degradation of apoproteins from HDL, but not for reverse cell membrane cholesterol transport. The activity of the binding site is up-regulated during cell proliferation and down-regulated during cell growth suppression.

Adult↗

PubMed1997

Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells.

Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest-derived ectomesenchyme follows its specific genetic control. The rat small eye (rSey) involves a mutation in the Pax-6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno-orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax-6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild-type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild-type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.

Animals↗

PubMed1996

Characterization of excitatory and inhibitory motor neurons to the guinea pig lower esophageal sphincter.

BACKGROUND & AIMS: The lower esophageal sphincter is innervated primarily by enteric motor neurons. The somata of excitatory and inhibitory motor neurons were identified and mapped. METHODS: Retrograde labeling in organotypic culture and immunohistochemistry were used to identify motor neuron somata. RESULTS: 1,1'-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (Dil) on the left side of the sphincter labeled descending motor neurons located up to 26 mm along the esophagus and locally (within 2 mm) and gastric motor neurons. Dil applied to the right side of the sphincter labeled descending and local motor neurons but very few gastric motor neurons. Inhibitory motor neuron cell bodies, identified by nitric oxide synthase immunoreactivity, accounted for 86% +/- 4% (n = 6) of descending motor neurons and 53% +/- 4% of local motor neurons labeled from the right side. Excitatory motor neurons, immunoreactive for choline acetyltransferase, accounted for 20% +/- 3% (n = 6) of descending motor neurons and for 47% +/- 4% of local motor neurons. All motor neurons were unipolar, but inhibitory motor neurons were significantly larger than excitatory neurons. CONCLUSIONS: The lower esophageal sphincter is innervated by local excitatory and inhibitory motor neurons and by descending esophageal inhibitory neurons. The oblique muscle, supplied by gastric motor neurons, is closely associated with the gastroesophageal junction.

Animals↗

PubMed1989

The effect of cyclosporin A on rat pancreatic B-cell: interaction with the calcium-antagonist diltiazem.

Cyclosporin A (CyA) was fed to male Wistar rats in a dose of 5, 10 or 50 mg/kg b.wt. for 7 days. In another experimental procedure 10 mg/kg b.wt. CyA was given for 7 days combined with Diltiazem (DIL) 6.25 mg/kg b.wt. once daily ip. Glucose stimulated insulin secretion from the isolated perfused pancreas was investigated in these animals. Total insulin secretion (ng/50 min) was not affected after feeding 5 mg/kg b.wt. CyA, but was significantly decreased with 10 and 50 mg/kg b.wt. CyA. The biphasic insulin secretion was reduced after 5 mg/kg b.wt. CyA during the initial peak (0-10 min) but not during the second peak (10-50 min), whereas after 10 and 50 mg/kg b.wt. CyA both peaks nearly reached base-line levels. DIL markedly increased the initial peak (0-10 min) after feeding CyA 10 mg/kg b.wt., whereas the second peak was not affected. Our results demonstrate a toxic effect of CyA on the pancreatic B-cell, which might be partly abolished by calcium-antagonists.

Animals↗

PubMed1985

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

Actins↗

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↗