102 results on '"Barisas, B. G."'
Search Results
2. Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells.
- Author
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Woodard, S L, primary, Aldo-Benson, M, additional, Roess, D A, additional, and Barisas, B G, additional
- Published
- 1995
- Full Text
- View/download PDF
3. Microcalorimetry of Biological Systems.
- Author
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Barisas, B G and Gill, S J
- Published
- 1978
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4. Molecular dynamics of point mutated I-Ak molecules expressed on lymphocytes
- Author
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Munnelly, H. M., Brady, C. J., Hagen, G. M., Horvat, R. D., Wade, W. F., Roess, D. A., and Barisas, B. G.
- Published
- 2001
- Full Text
- View/download PDF
5. Rotational and lateral dynamics of I-A(k) molecules expressing cytoplasmic truncations.
- Author
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Munnelly, H M, Brady, C J, Hagen, G M, Wade, W F, Roess, D A, and Barisas, B G
- Abstract
Rotational and lateral diffusion of I-A(k) molecules with various alpha and beta chain cytoplasmic truncations known to affect class II function were measured to assess the role of cytoplasmic domains in regulating I-A(k) molecular motions. Deletion of all 12 alpha chain C-terminal residues and all 18 corresponding beta chain residues (alpha-12/beta-18) is known to abrogate translocation of protein kinase C to the nucleus upon class II cross-linking. Similarly, truncation of the entire cytoplasmic alpha chain domain and the 10 C-terminal residues of the beta chain impairs presentation of antigenic peptides to T cells. The rotational correlation time of the wild-type molecule, 11.9 +/- 2.6 micros as measured by time-resolved phosphorescence anisotropy, decreased to 7. 2 +/- 3.7 micros in the fully truncated alpha-12/beta-18 protein. Other truncated class II molecules exhibited only small changes in molecular rotation rates relative to the wild-type. The rate of lateral diffusion of the fully truncated molecule, measured with two independent methods, 2.3 x 10(-10) cm(2)/s, was comparable with that of the wild-type molecule. Thus, it appears that the alpha and beta chain cytoplasmic domains regulate the molecular motions of unperturbed I-A(k) molecules only modestly, despite the known involvement of these regions in class II signaling. Various explanations for this behavior are discussed, e.g. the possibility that class II membrane complexes are sufficiently large that association and dissociation of specific signaling proteins during antigen presentation do not significantly perturb the apparent molecular motions of the complex.
- Published
- 2000
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- View/download PDF
6. Mutations in specific I-A(k) alpha(2) and beta(2) domain residues affect surface expression.
- Author
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Lang, M L, Yadati, S, Seeley, E S, Nydam, T, Wade, T K, Gabriel, J L, Yeaman, G, Barisas, B G, and Wade, W F
- Abstract
A previous investigation demonstrated that several mutations in class II dimer-of-dimers contact residues interfere with antigen presentation by transfectants but not with plasma membrane expression of the mutant class II. In the present study we examined other class II mutations in this region that did inhibit plasma membrane expression of mutant class II molecules. Molecules containing both mutations H alpha 181D in the alpha(2) domain and E beta 170K in the beta(2) domain exhibited low plasma membrane expression, but molecules with only one of these mutations were expressed normally. The mutant class II molecules were transported to organelles that were accessible to a fluid-phase protein, hen egg lysozyme (HEL). Culture of transfectants with lysozyme enhanced the amount of class II compact dimer (alpha beta plus peptide; CD), and this was especially marked for the class II mutant H alpha 181D/E beta 170K and for other molecules possessing both mutations. Formation of class II CD was not paralleled by an increase in class II surface expression. Thus the joint mutation of H alpha 181 and E beta 170 has two effects. In the absence o high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting invariant chain (Ii) proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not expression of class II on the plasma membrane. Molecular modeling of the possible interaction of class II and Ii suggests that the mutant amino acids H alpha 181D and E beta 170K, besides affecting the overall stability of class II, might also interact with Ii via two loops in class II's alpha(2) and beta(2) domains respectively.
- Published
- 2000
- Full Text
- View/download PDF
7. Salt bridge residues between I-A^k dimer of dimers a-chains modulate antigen presentation
- Author
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Yadati, S., Nydam, T., Demian, D., Wade, T. K., Gabriel, J. L., Barisas, B. G., and Wade, W. F.
- Published
- 1999
- Full Text
- View/download PDF
8. Calorimetric studies of inositol hexaphosphate binding to aquomethemoglobin A
- Author
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Barisas, B. G. and Barton, D.
- Published
- 1998
- Full Text
- View/download PDF
9. Mutations in MHC class II dimer of dimers contact residues: effects on antigen presentation.
- Author
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Nydam, T, Wade, T K, Yadati, S, Gabriel, J L, Barisas, B G, and Wade, W F
- Abstract
The recent solutions of the MHC class II crystal structure reveal dimerization of the alphabeta heterodimers. These dimer of dimers structures may also exist either on resting cells or after engagement by TCR, and may be involved in B cell signaling and up-regulation of co-stimulatory molecules such as B7 which facilitate T cell activation. By combining crystallographic data on HLA-DR1 with the sequence of murine I-Ak and refining the resulting structure through energy minimization calculations, we have predicted the contact amino acids expected to stabilize the I-Ak dimer of dimers structure. As in HLA-DR1, three salt bridges in I-Ak (D alpha62-Hbeta112, H alpha181-E beta163, E alpha183-Hbeta113) appear to provide the main interaction. Guided by this structural data, we prepared 45 B cell transfectants representing 20 different class II mutation phenotypes in the contact region containing these salt bridges. We examined their abilities to activate three T cell hybrids. Antigen-specific h4Ly50.5 cells were not greatly affected by changes in the dimer of dimer contact residues. In contrast, autoreactive C8.A3 T cells were very sensitive to changes in this region but presentation of class II of many mutation phenotypes could be rescued by treatments that up-regulate B7-1. The alloreactive hybridoma 2H40.2.5 was less sensitive to changes in the contact residues. A simple model was developed that summarizes the effects of the mutations for the T cells tested. Mutations at D alpha162, E alpha183, H alpha181 and Rbeta106 had the largest negative impact, while D alpha166, E alpha185, Hbeta112, Hbeta113 and E beta163 were less disruptive. Results are consistent with mutations interfering with class II interaction with another molecule which might or might not be another class II heterodimer. However, the larger negative impact of alpha chain mutations in salt bridge pairs suggests that these sites also help maintain some essential conformation of the alpha chain apart from any possible impact on dimer of dimers stability.
- Published
- 1998
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10. Herpes simplex virus binding and entry modulate cell surface protein mobility
- Author
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Rosenthal, K S, Leuther, M D, and Barisas, B G
- Abstract
Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.
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- 1984
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11. Lateral dynamics of major histocompatibility complex Class II molecules bound with agonist peptide or altered peptide ligands
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Qiu, Y., Wade, W. F., Roess, D. A., and Barisas, B. G.
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- 1996
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12. Thermodynamics of hapten binding to MOPC [mouse plasmacytoma] 315 and MOPC 460 mouse myeloma proteins
- Author
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Johnston, M. F. M., primary, Barisas, B. G., additional, and Sturtevant, J. M., additional
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- 1974
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13. Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin.
- Author
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Peacock, J S, primary and Barisas, B G, additional
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- 1981
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14. Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes.
- Author
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Londo, T R, primary, Peacock, J S, additional, Roess, D A, additional, and Barisas, B G, additional
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- 1986
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15. Differences in the Lateral Mobility of Receptors for Luteinizing Hormone (LH) in the Luteal Cell Plasma Membrane when Occupied by Ovine LH Versus Human Chorionic Gonadotropin*
- Author
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NISWENDER, G. D., primary, ROESS, D. A., additional, SAWYER, H. R., additional, SILVIA, W. J., additional, and BARISAS, B. G., additional
- Published
- 1985
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16. Changes in lectin receptor lateral mobilities accompany lymphocyte stimulation.
- Author
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Leuther, M D, primary, Peacock, J S, additional, Krakauer, H, additional, and Barisas, B G, additional
- Published
- 1981
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17. Heats of carbon monoxide binding by hemoglobin M Iwate
- Author
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Gaud, H. T., primary, Gill, S. J., additional, Barisas, B. G., additional, and Gersonde, K., additional
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- 1975
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18. Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.
- Author
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Peacock, J S, primary, Londo, T R, additional, Roess, D A, additional, and Barisas, B G, additional
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- 1986
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19. Photobleaching recovery studies of T-independent antigen mobility on antibody-bearing liposomes.
- Author
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Peacock, J S, primary and Barisas, B G, additional
- Published
- 1983
- Full Text
- View/download PDF
20. Cytochalasins and Colchicine Increase the Lateral Mobility of Human Chorionic Gonadotropin-Occupied Luteinizing Hormone Receptors on Ovine Luteal Cells*
- Author
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ROESS, D. A., primary, NISWENDER, G. D., additional, and BARISAS, B. G., additional
- Published
- 1988
- Full Text
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21. Thermodynamics of the binding of haptens to rabbit anti-2,4-dinitrophenyl antibodies
- Author
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Sturtevant, J. M., primary, Barisas, B. G., additional, and Singer, S. J., additional
- Published
- 1971
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22. Thermodynamics of the binding of 2,4-dinitrophenyl and 2,4,6-trinitrophenyl haptens to the homologous and heterologous rabbit antibodies
- Author
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Barisas, B. G., primary, Singer, S. J., additional, and Sturtevant, J. M., additional
- Published
- 1972
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23. Fluorescence depletion measurements in various experimental geometries provide true emission and absorption anisotropies for the study of protein rotation
- Author
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Londo, T. R., Rahman, N. A., Roess, D. A., and Barisas, B. G.
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- 1993
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24. Effect of fish meal supplementation on spatial distribution of lipid microdomains and on the lateral mobility of membrane-bound prostaglandin F 2α receptors in bovine corpora lutea.
- Author
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Plewes MR, Burns PD, Graham PE, Bruemmer JE, Engle TE, and Barisas BG
- Subjects
- Animal Nutritional Physiological Phenomena, Animals, Corpus Luteum cytology, Corpus Luteum drug effects, Diet veterinary, Female, Receptors, Prostaglandin genetics, Animal Feed analysis, Cattle, Dietary Supplements, Fish Products, Lipid Metabolism drug effects, Receptors, Prostaglandin metabolism
- Abstract
This study examined the effects of fish meal supplementation on spatial distribution of lipid microdomains and lateral mobility of prostaglandin F
2α (FP) receptors on cell plasma membranes of the bovine corpus luteum (CL). Beef cows were stratified by BW and randomly assigned to receive a corn gluten meal supplement (n = 4) or fish meal supplement (n = 4) for 60 d to allow incorporation of fish meal-derived omega-3 fatty acids into luteal tissue. Ovaries bearing the CL were surgically removed between days 10 to 12 after estrus corresponding to approximately day 60 of supplementation. A 200-mg sample of luteal tissue was analyzed for fatty acid content using gas-liquid chromatography (GLC). The remaining tissue was enzymatically digested with collagenase to dissociate individual cells from the tissue. Cells were cultured to determine the effects of dietary supplementation on lipid microdomains and lateral mobility of FP receptors. Luteal tissue collected from fish meal-supplemented cows had increased omega-3 fatty acids content (P < 0.05). Lipid microdomain total fluorescent intensity was decreased in dissociated luteal cells from fish meal-supplemented cows (P < 0.05). Micro and macro diffusion coefficients of FP receptors were greater for cells obtained from fish meal-supplemented cows (P < 0.05). In addition, compartment diameter of domains was larger, whereas resident time was shorter for receptors from cells obtained from fish meal-supplemented cows (P < 0.05). Data indicate that dietary supplementation with fish meal increases omega-3 fatty acid content in luteal tissue causing disruption of lipid microdomains. This disruption leads to increased lateral mobility of the FP receptor, increased compartment sizes, and decreased resident time, which may influence prostaglandin signaling in the bovine CL., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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- View/download PDF
25. Effect of fish oil on lateral mobility of prostaglandin F 2α (FP) receptors and spatial distribution of lipid microdomains in bovine luteal cell plasma membrane in vitro.
- Author
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Plewes MR, Burns PD, Graham PE, Hyslop RM, and Barisas BG
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- Animals, Cell Membrane chemistry, Cell Membrane metabolism, Cells, Cultured, Diffusion drug effects, Dinoprost pharmacology, Female, Cattle, Cell Membrane drug effects, Fish Oils pharmacology, Lipids analysis, Luteal Cells ultrastructure, Receptors, Prostaglandin metabolism
- Abstract
Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F
2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P < 0.01). In experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger, and residence times were reduced for receptors in fish oil-treated cells (P < 0.05). In experiment 3, single particle tracking was used to determine the effects of PGF2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil-treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF2α ., (Copyright © 2016 Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
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26. Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET).
- Author
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Lidke DS, Nagy P, Barisas BG, Heintzmann R, Post JN, Lidke KA, Clayton AH, Arndt-Jovin DJ, and Jovin TM
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- Animals, CHO Cells, Cell Line, Cricetinae, Dose-Response Relationship, Drug, ErbB Receptors chemistry, Flow Cytometry, Green Fluorescent Proteins, Humans, Luminescent Proteins metabolism, Microscopy, Confocal, Models, Statistical, Mutation, Anisotropy, Microscopy, Fluorescence instrumentation, Microscopy, Fluorescence methods
- Abstract
We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.
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- 2003
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27. Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors.
- Author
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Horvat RD, Roess DA, Nelson SE, Barisas BG, and Clay CM
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- Animals, Bacterial Proteins chemistry, CHO Cells, Cricetinae, Cyclic AMP biosynthesis, Cyclic AMP metabolism, Diffusion, Gonadotropin-Releasing Hormone analogs & derivatives, Gonadotropin-Releasing Hormone metabolism, Green Fluorescent Proteins, Hormone Antagonists metabolism, Kinetics, Luminescent Proteins chemistry, Oligopeptides metabolism, Receptors, LHRH agonists, Receptors, LHRH antagonists & inhibitors, Recombinant Fusion Proteins chemistry, Signal Transduction physiology, Spectrometry, Fluorescence, Gonadotropin-Releasing Hormone pharmacology, Hormone Antagonists pharmacology, Oligopeptides pharmacology, Receptors, LHRH physiology
- Abstract
We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.
- Published
- 2001
- Full Text
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28. Luteinizing hormone receptors are self-associated in slowly diffusing complexes during receptor desensitization.
- Author
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Horvat RD, Barisas BG, and Roess DA
- Subjects
- Animals, Bacterial Proteins genetics, Bacterial Proteins metabolism, CHO Cells, Cell Membrane metabolism, Chorionic Gonadotropin pharmacology, Cricetinae, Cyclic AMP metabolism, Energy Transfer, Fluorescence, Fluorescent Dyes chemistry, Green Fluorescent Proteins, Humans, Luminescent Proteins genetics, Luminescent Proteins metabolism, Luteinizing Hormone pharmacology, Rats, Receptors, LH drug effects, Receptors, LH genetics, Rhodamines chemistry, Sheep, Chorionic Gonadotropin metabolism, Luteinizing Hormone metabolism, Receptors, LH metabolism
- Abstract
We have previously shown that rat LH receptors (LHRs) occupied by human CG (hCG) exhibit slow receptor lateral diffusion and are self-associated. Here we have examined whether LHRs become self-associated and enter slowly diffusing structures in response to hormone binding and whether these receptors retain this organization while in the desensitized state. Before hormone exposure, wild-type rat LHRs coupled at the C terminus to enhanced green fluorescent protein (GFP-LHR-wt) exhibited fast lateral diffusion, as assessed by fluorescent photobleaching recovery (FPR) methods, and most receptors were laterally mobile. After 30 min exposure to hCG and subsequent removal of hormone by low pH wash, hormone challenge at any time within the next 4 h produced no increase in cellular cAMP levels. During this time, LHRs were either laterally immobile or exhibited slower lateral diffusion. When LHRs were again responsive to binding of hormone, the rate of receptor lateral diffusion had become significantly faster and the fraction of mobile receptors was again large. Desensitized LHRs were also self-associated and present in microscopically visible clusters on the plasma membrane. Fluorescence energy transfer (FET) methods were used to measure the extent of interaction between receptors coupled to either GFP or to yellow fluorescent protein (YFP). Before hormone treatment, there was essentially no energy transfer between LHRs. After desensitization of the receptors by 30 min exposure to hCG, energy transfer efficiency increased to 18%. Values for FET efficiency between desensitized receptors decreased over time, and receptors were responsive to hormone only after measurable energy transfer had completely disappeared. Together these results suggest that desensitized LHRs exist in large, slowly diffusing structures containing self-associated receptors and that these structures must dissipate before the receptor can again respond to hormone.
- Published
- 2001
- Full Text
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29. Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane.
- Author
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West LA, Horvat RD, Roess DA, Barisas BG, Juengel JL, and Niswender GD
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- Animals, COS Cells, Chlorocebus aethiops, Kinetics, Membrane Proteins metabolism, Recombinant Fusion Proteins metabolism, Sheep, Transfection, Intracellular Membranes metabolism, Mitochondria metabolism, Phosphoproteins metabolism, Receptors, GABA-A metabolism
- Abstract
Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have both been implicated in the transport of cholesterol across mitochondrial membranes in steroidogenic cells. Therefore, we hypothesized that StAR and PBR were associated in this process. To test this hypothesis, we measured fluorescence energy transfer (FET) between these proteins by fusing enhanced green fluorescent protein (GFP, donor fluorophore) and yellow fluorescent protein (YFP, acceptor fluorophore) to the C-terminus of ovine StAR (37GFP) and ovine PBR (PBRYFP), respectively. These intrinsically fluorescent proteins were stably transfected into Cos-7 cells and determined to be biologically active. For FET to occur the appropriate fluorescent molecules need to be <100 A from each other. We observed 22.0 +/- 0.9% energy transfer efficiency for 37GFP and PBRYFP, a 4.9 fold increase above non-specific energy transfer between free GFP and PBRYFP (p <.0001). Thus, it appears that StAR and PBR are closely associated in mitochondrial membranes and that these molecules may interact in the transportation of cholesterol.
- Published
- 2001
- Full Text
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30. Luteinizing hormone receptors are self-associated in the plasma membrane.
- Author
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Roess DA, Horvat RD, Munnelly H, and Barisas BG
- Subjects
- Animals, Cells, Cultured, Chemical Phenomena, Chemistry, Physical, Chorionic Gonadotropin metabolism, Diffusion, Dimerization, Energy Transfer, Fluorescein-5-isothiocyanate, Fluorescence, Fluorescent Dyes, Gene Expression, Humans, Luteinizing Hormone metabolism, Microscopy, Fluorescence, Photochemistry, Rats, Receptors, LH chemistry, Receptors, LH genetics, Rhodamines, Structure-Activity Relationship, Transfection, Cell Membrane metabolism, Receptors, LH metabolism
- Abstract
We have evaluated rat LH receptor self-association and lateral dynamics for functional and nonfunctional receptors after binding of hormone. We demonstrate, for the first time, that grouped receptors observed in electron or light microscopy represent actual receptor dimers or oligomers rather than simply the concentration of receptors within membrane microdomains. Fringe fluorescence photobleaching recovery methods showed that, after binding of either LH or human CG (hCG), functional wild-type LH receptors, expressed on 293 cells (LHR-wt cells), have mobilities that are 25% lower than those of nonfunctional LH receptors containing an arginine substitution for lysine at position 583 (LHR-K583R cells). Because lateral diffusion coefficients in two dimensions depend only on the logarithm of the molecular size of the diffusing species, this result implies that functional receptors exist in substantially larger membrane complexes than do nonfunctional receptors. In single-cell measurements of fluorescence energy transfer after hormone binding, functional LH receptors were also characterized by receptor self-aggregation. Values for fluorescence resonant energy transfer efficiency were 13 +/- 2% and 17 +/- 3% between fluorophore-conjugated LH or hCG, respectively, bound to receptors on LHR-wt cells. However, there was little or no energy transfer between receptors on LHR-K583R cells. These results suggest that receptor functionality involves receptor-receptor interactions and that the extent of such receptor self-association depends on whether LH or hCG binds the receptor.
- Published
- 2000
- Full Text
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31. Förster distances between green fluorescent protein pairs.
- Author
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Patterson GH, Piston DW, and Barisas BG
- Subjects
- Green Fluorescent Proteins, Luminescent Proteins genetics, Luminescent Proteins isolation & purification, Spectrometry, Fluorescence, Spectrophotometry, Ultraviolet, Luminescent Proteins chemistry
- Published
- 2000
- Full Text
- View/download PDF
32. Biological function of the LH receptor is associated with slow receptor rotational diffusion.
- Author
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Roess DA, Brady CJ, and Barisas BG
- Subjects
- Animals, Anisotropy, Cell Line, Cyclic AMP metabolism, Diffusion, Erythrosine, Gene Expression, Humans, Kinetics, Luminescent Measurements, Luteinizing Hormone metabolism, Mutation, Rats, Receptors, LH chemistry, Receptors, LH genetics, Rotation, Structure-Activity Relationship, Temperature, Transfection, Chorionic Gonadotropin metabolism, Receptors, LH metabolism
- Abstract
The biological activity of luteinizing hormone (LH) receptors can be affected by modifications to the receptor's amino acid sequence or by binding of hormone antagonists such as deglycosylated hCG. Here we have compared rotational diffusion of LH receptors capable of activating adenylate cyclase with that of non-functional hormone-occupied receptors at 4 degrees C and 37 degrees C using time-resolved phosphorescence anisotropy techniques. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) or to the LH receptor on MA-10 cells produces functional receptors which exhibit rotational correlation times longer than 1000 micros. However, modification of the LH receptor by substitution of Lys583-->Arg (LHR-K583R) results in a receptor that is non-functional and which has a significantly shorter rotational correlation time of 130+/-12 micros following binding of hCG. When these receptors are treated with deglycosylated hCG, an inactive form of hCG, the rotational correlation times for the LH receptors on LHR-wt and MA-10 cells are also shorter, namely 64+/-8 and 76+/-14 micros, respectively. Finally, a biologically active truncated form of the rat LH receptor expressed in 293 cells (LHR-t631) has slow rotational diffusion, greater than 1000 micros, when occupied by hCG and a significantly shorter rotational correlation time of 103+/-12 micros when occupied by deglycosylated hCG. The effects of rat LH binding to LH receptors on these various cell lines were similar to those of hCG although the magnitude of the changes in receptor rotational diffusion were less pronounced. We suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for non-functional hormone-receptor complexes may reflect the absence of essential interactions between these complexes and other membrane proteins.
- Published
- 2000
- Full Text
- View/download PDF
33. Dynamics of molecules involved in antigen presentation: effects of fixation.
- Author
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Barisas BG, Wade WF, Jovin TM, Arndt-Jovin D, and Roess DA
- Subjects
- Animals, Anisotropy, Antigen-Presenting Cells immunology, Cell Line, Diffusion, Fixatives, Formaldehyde, Histocompatibility Antigens Class II chemistry, Luminescent Measurements, Mice, Polymers, Antigen Presentation, Histocompatibility Antigens Class II metabolism
- Abstract
Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-Ad on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-Ad at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 micros unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from approximately 2x10(-10) cm2 s(-1), while the fractional recovery remained unchanged at about 40-50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.
- Published
- 1999
- Full Text
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34. Intrinsically fluorescent luteinizing hormone receptor demonstrates hormone-driven aggregation.
- Author
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Horvat RD, Nelson S, Clay CM, Barisas BG, and Roess DA
- Subjects
- Animals, CHO Cells, Cell Membrane chemistry, Cell Membrane metabolism, Chorionic Gonadotropin metabolism, Cricetinae, Cyclic AMP biosynthesis, Green Fluorescent Proteins, Humans, Luminescent Proteins biosynthesis, Luminescent Proteins genetics, Microscopy, Confocal, Microscopy, Fluorescence, Receptors, LH biosynthesis, Receptors, LH genetics, Recombinant Fusion Proteins biosynthesis, Luminescent Proteins metabolism, Receptor Aggregation, Receptors, LH metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism
- Abstract
The possibility that LH receptors exist as isolated molecules when unbound and aggregate upon binding gonadotropins has previously been untestable in viable cells for want of a suitable nonhormone probe. We have now expressed in CHO cells an intrinsically-fluorescent LH receptor involving enhanced green fluorescent protein (GFP) fused to the C-terminus of the rat LH receptor (rLHR-GFP). More than half of these receptors (54 +/- 4%) are located on the plasma membrane and are functional: cAMP levels increase 3-5 fold in response to 10 nM LH or hCG. In fluorescence photobleaching recovery studies at 37 degrees C, 54 +/- 13% of unoccupied rLHR-GFP were laterally mobile with a diffusion coefficient D of 16 +/- 3.5 x 10(-10)cm2sec-1. Introduction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 +/- 1.3 x 10(-10)cm2sec-1 and reduced fluorescence recovery after photobleaching to 27 +/- 1%. Following treatment with 1 nM hCG, rLHR-GFP were laterally immobile and were distributed into small fluorescent patches over the cell surface. Thus, unoccupied rLHR-GFP receptors apparently exist as dispersed plasma membrane proteins with comparatively fast lateral diffusion. Interaction of receptors with LH or hCG caused clustering of rLHR-GFP receptors, significantly restricting lateral diffusion., (Copyright 1999 Academic Press.)
- Published
- 1999
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35. Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion.
- Author
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Nelson S, Horvat RD, Malvey J, Roess DA, Barisas BG, and Clay CM
- Subjects
- Animals, Binding, Competitive, CHO Cells, COS Cells, Cell Membrane metabolism, Cricetinae, Diffusion, Gonadotropin-Releasing Hormone analogs & derivatives, Gonadotropin-Releasing Hormone metabolism, Green Fluorescent Proteins, Indicators and Reagents, Ligands, Luminescent Proteins genetics, Mice, Receptors, LHRH genetics, Receptors, LHRH metabolism, Recombinant Fusion Proteins metabolism, Receptors, LHRH physiology
- Abstract
The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.
- Published
- 1999
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36. Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.
- Author
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Munnelly HM, Roess DA, Wade WF, and Barisas BG
- Subjects
- Animals, Cardiolipins, Cell Line, Cholesterol, Diffusion, Equipment Design, Histocompatibility Antigens Class II biosynthesis, Kinetics, Liposomes, Mice, Microscopy, Interference instrumentation, Microscopy, Interference methods, Models, Theoretical, Phosphatidylcholines, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Reproducibility of Results, Sensitivity and Specificity, Spectrometry, Fluorescence instrumentation, Spectrometry, Fluorescence methods, Transfection, Histocompatibility Antigens Class II chemistry
- Abstract
Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5 microm 1/e2 radius. The effective spot area represents only 1/500 of the total surface of an 8-microm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100 molecules. This limits the precision and reproducibility of FPR measurements. We describe a method for interferometric fringe pattern FPR that permits simultaneous interrogation of the entire cell's surface. Fringe patterns are generated interferometrically within the optical path of an FPR system. Methods for interpreting fluorescence recovery kinetics on cells and for determining the protein mobile fraction are presented. With fringe FPR, the murine major histocompatibility complex class II antigen I-Ak expressed on M12.C3.F6 cells has 100-fold improved fluorescence signals relative to spot FPR, with corresponding improvements in signal-to-noise ratios of recovery traces. Diffusion coefficients (+/- standard deviation) of (2.1 +/- 0.4) x 10(-10) and (1.8 +/- 1.0) x 10(-10) cm2 s-1 with corresponding mobile fractions of I-Ak of 66.1 +/- 7.8% and 63.4 +/- 18.0% were obtained by fringe and spot methods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be substantial variation from cell to cell in protein dynamics, and this method may permit the assessment of such variation.
- Published
- 1998
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37. Luteinizing hormone receptors are associated with non-receptor plasma membrane proteins on bovine luteal cell membranes.
- Author
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Roess DA, Rahman NA, Munnelly H, Meiklejohn BI, Brady CJ, and Barisas BG
- Subjects
- Animals, Azides, Cattle, Cell Membrane chemistry, Chorionic Gonadotropin, Energy Transfer, Female, Fluorescent Dyes, Humans, Iodine Radioisotopes, Luteinizing Hormone, Membrane Proteins chemistry, Molecular Weight, Sheep, Corpus Luteum chemistry, Membrane Proteins analysis, Receptors, LH chemistry
- Abstract
Biophysical studies of the bovine luteinizing hormone (LH) receptor on luteal cell membranes suggest that this receptor may be part of a larger molecular weight structure. We have used 5-iodonaphthyl-1-azide (INA) to identify plasma membrane proteins near LH receptors on plasma membranes from bovine corpora lutea. Following binding of eosin isothiocyanate-derivatized ovine LH or human chorionic gonadotropin (hCG), five proteins with molecular weights of 71, 57, 55, 49 and 36 kDa were selectively derivatized with [125I]-INA following 2 h exposure at 22 degreesC to 514 nm light. However, there was no fluorescence energy transfer between LH receptors occupied by ovine LH or hCG indicating that LH receptors were not self-associated in these membrane preparations. Together these results suggest that, following hormone binding, single copies of the LH receptor may exist in large molecular weight structures that include non-receptor proteins., (Copyright 1998 Elsevier Science B.V.)
- Published
- 1998
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38. The rotational diffusion of LH receptors differs when receptors are occupied by hCG versus LH and is increased by cytochalasin D.
- Author
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Roess DA, Jewell MA, Philpott CJ, and Barisas BG
- Subjects
- Actin Cytoskeleton drug effects, Animals, Cell Line, Cell Membrane metabolism, Colchicine pharmacology, Cytochalasin B pharmacology, Diffusion, Fluorescence Polarization, Leydig Cells metabolism, Male, Microtubules drug effects, Receptors, LH chemistry, Receptors, LH metabolism, Temperature, Chorionic Gonadotropin metabolism, Cytochalasin D pharmacology, Luteinizing Hormone metabolism, Receptors, LH drug effects
- Abstract
We have examined the rotational diffusion of the luteinizing hormone (LH) receptors binding human chorionic gonadotropin (hCG) or ovine luteinizing hormone (oLH) in MA-10 Leydig tumor cells using time-resolved phosphorescence anisotropy techniques. LH receptors binding erythrosin isothiocyanate (ErITC)-derivatized oLH were rotationally mobile with rotational correlation times of 62 micros, 48 micros, 38 micros, and 29 micros at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. ErITC-hCG bound to the LH receptor was rotationally immobile, showing no anisotropy decay at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C. To determine whether cytoskeletal components influenced the rotational diffusion of LH receptors, we measured rotational diffusion of LH receptors on MA-10 cells treated with 20 microg/ml cytochalasin D and on plasma membrane preparations. Following 1 h exposure to cytochalasin D, the rotational correlation times for hCG-occupied LH receptors were typically 11 micros at 37 degrees C compared to > 1000 micros on untreated cells. Treatment of MA-10 cells with cytochalasin B or colchicine had no affect on LH receptor rotational diffusion. Rotational correlation times for LH-occupied receptors decreased from 29 micros to 12 micros at 37 degrees C following cytochalasin D treatment. The rotational diffusion of LH receptors on plasma membrane preparations was similar to that observed for LH- and hCG-occupied receptors on intact cells treated with cytochalasin D. These various results indicate that there are differential effects of LH and hCG binding on the interactions of LH receptors with plasma membrane proteins and that microfilaments anchor the hCG- and LH-occupied receptors.
- Published
- 1997
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39. 5-iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer.
- Author
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Meiklejohn BI, Rahman NA, Roess DA, and Barisas BG
- Subjects
- Animals, B-Lymphocytes, Blood Proteins chemistry, Energy Transfer, Eosine Yellowish-(YS), Erythrocyte Membrane, Female, Fluorescein, Fluoresceins, Humans, Immunoglobulin mu-Chains chemistry, Leukemia, Basophilic, Acute, Membrane Proteins analysis, Mice, Mice, Inbred BALB C, Photosensitizing Agents, Rats, Tumor Cells, Cultured, Azides, Cross-Linking Reagents, Membrane Proteins chemistry, Receptors, IgE chemistry
- Abstract
We have examined conditions optimal for 5-iodonaphthyl-1-azide (INA4) labeling of membrane proteins proximal to known membrane sites. Membrane-bound INA can be indirectly activated by energy transfer from visible chromophores. We demonstrate that the efficiency of this sensitized activation is enhanced by use of triplet-forming chromophores such as eosin and by deoxygenation. Variation of sensitized activation efficiency with INA concentration indicates that the critical distance for eosin-INA energy transfer in solution is 8-14 A. We suggest that photosensitization occurs through triplet exchange and present an improved labeling protocol based on these findings. This protocol was used to examine whether different accessory proteins are associated with isolated and crosslinked Type I Fc epsilon receptors on 2H3 rat basophilic leukemia cells. 2H3 cells were incubated with eosin-conjugated IgE and irradiated at 514 nm yielding [125I]INA derivatized peptides at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE prior to irradiation labeled three additional proteins at 60, 54, and 43 kDa. These results demonstrate the utility of sensitized INA labeling in characterizing protein-protein interactions in membranes of intact cells and indicate the importance of considering photophysical factors when selecting sensitizers and reaction conditions. We discuss estimation of the size of the membrane region surrounding a sensitizing chromophore within which INA labeling of membrane proteins occurs.
- Published
- 1997
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40. Rotational dynamics of luteinizing hormone receptors on bovine and ovine luteal cell plasma membranes.
- Author
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Philpot CJ, Rahman NA, Kenny N, Barisas BG, and Roess DA
- Subjects
- Animals, Anisotropy, Cattle, Cell Membrane chemistry, Cell Membrane ultrastructure, Chorionic Gonadotropin metabolism, Corpus Luteum ultrastructure, Diffusion, Epidermal Growth Factor metabolism, Erythrosine pharmacology, Female, Sheep, Species Specificity, Temperature, Corpus Luteum chemistry, Receptors, LH chemistry
- Abstract
To determine whether LH receptor rotational diffusion is similar in closely related species, we compared the rotational correlation times of LH receptors on bovine CL membranes with those of LH receptors on sheep small luteal cells and luteal cell plasma membranes using time-resolved phosphorescence anisotropy techniques. After binding of erythrosin isothiocyanate (ErITC)-derived bovine LH (bLH), ErITC-ovine LH (oLH), or ErITC-hCG, there was no difference in the initial and final anisotropy at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, indicating that the bLH receptor was rotationally immobile on the time scale of our experiments. On these same membrane preparations, the epidermal growth factor (EGF) receptor occupied by ErITC-murine EGF exhibited temperature-dependent rotational correlation times of 80 +/- 5 microseconds, 111 +/- 7 microseconds, 254 +/- 4 microseconds, and > 1000 microseconds at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. Slower rotational times for EGF receptor observed at higher temperatures suggested the occurrence of temperature-dependent receptor aggregation. Like the bLH receptor, the oLH receptor on intact cells and on CL plasma membranes was rotationally immobile on the time scale of our experiments when occupied by ErITC-hCG. However, the oLH-occupied receptors on small luteal cells and on luteal cell membranes had comparable rotational correlation times at 37 degrees C. These results suggest that bLH receptors are present in large, rotationally immobile structures, whereas the receptor-containing structure formed on ovine luteal cells depends on whether that receptor is occupied by hCG or oLH. Also, despite the similarities between reproductive function in these species, the LH-occupied receptor appears to be organized differently in the plasma membranes of these hormone-responsive luteal cells.
- Published
- 1995
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41. Rotational dynamics of luteinizing hormone receptors and MHC class I antigens on murine Leydig cells.
- Author
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Philpott CJ, Rahman NA, Kenny N, Londo TR, Young RM, Barisas BG, and Roess DA
- Subjects
- Animals, Cell Membrane physiology, Chorionic Gonadotropin, Erythrosine, Fluorescence Polarization, Luteinizing Hormone, Male, Mice, Mice, Inbred BALB C, Histocompatibility Antigens Class I physiology, Leydig Cells physiology, Receptors, LH physiology
- Abstract
We have examined the molecular motions of luteinizing hormone (LH) receptor and the Major Histocompatibility Complex Class I antigen on murine Leydig cells. Using time-resolved phosphorescence anisotropy methods, erythrosin (ErITC)-derivatized ovine luteinizing hormone (oLH) bound to the LH receptor appears rotationally mobile with rotational correlation times of 19.6 +/- 1.3 microseconds, 13.3 +/- 2.4 microseconds, 9.5 +/- 0.7 microseconds and 4.7 +/- 0.5 microseconds at 4 degrees C, 15 degrees C, 25 degrees C and 37 degrees C, respectively. Rotational correlation times for human chorionic gonadotropin (hCG)-occupied LH receptors were similar to those of the ErITC-oLH occupied receptor at each temperature. In addition, both oLH- and hCG-occupied LH receptors were laterally mobile in fluorescence photobleaching recovery experiments with diffusion coefficients at 29 degrees C of (5.8 +/- 0.9) x 10(-10) cm2 s-1 and (2.9 +/- 0.4) x 10(-10) cm2 s-1, respectively. We also measured the rotational correlation time of Class I antigen on murine Leydig cells using ErITC-derivatized 34-12-2S, an anti-Class I monoclonal antibody. Because there was no decay of the anisotropy function at 4 degrees C, 15 degrees C, 25 degrees C or 37 degrees C in the absence of oLH or following preincubation of Leydig cells with 1 nM oLH, it appears that Class I is rotationally immobile on the 1 ms timescale of our experiments. This result is consistent with the presence of Class I antigen in large molecular weight structures and may be the result of Class I self-aggregation. Further, treatment of cells with anti-Class I antibody had no effect on either basal or oLH-stimulated testosterone secretion. Thus, it appears that this anti-Class I antibody is not LH-mimetic on murine Leydig cells.
- Published
- 1995
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42. Truncation of the A alpha chain of MHC class II molecules results in inefficient antigen presentation to antigen-specific T cells.
- Author
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Wade WF, Ward ED, Rosloniec EF, Barisas BG, and Freed JH
- Subjects
- Animals, Antigen Presentation genetics, Egg Proteins immunology, Fixatives pharmacology, Flow Cytometry, Histocompatibility Antigens Class II genetics, Hybridomas immunology, Intercellular Adhesion Molecule-1 immunology, Lymphocyte Function-Associated Antigen-1 immunology, Mice, Muramidase immunology, Structure-Activity Relationship, Antigen Presentation physiology, Histocompatibility Antigens Class II chemistry, T-Lymphocytes immunology
- Abstract
Antigen presenting cells (APC) expressing MHC class II molecules composed of chains with part or all of the cytoplasmic domains deleted are inefficient at presenting hen egg lysozyme peptides to antigen specific T cell hybrids compared with APC that express wild-type MHC class II molecules. This effect is most apparent for mutants in which the alpha chain has been truncated. The inefficiency in antigen presentation can be amplified by pulsing the APC for 4 h with peptide rather than having peptide present throughout the presentation assay. Fixation of antigen-pulsed APC improves the capacity of APC with truncated class II molecules to stimulate T cell hybrids. Fixation of APC prior to exposure to antigen also leads to significant improvement in antigen presentation by the truncated class II molecules. Because the inefficiency of a given hybrid for antigen presentation does not correlate with its ability to transduce a signal as measured by protein kinase C translocation, we suggest that defects in this pathway are not the only cause of impaired antigen presentation. However, because previous studies have demonstrated the need for an intact cytoskeleton for successful antigen presentation, we propose that the carboxy truncated class II molecules are inefficient in antigen presentation because they are unable to generate the signal that ultimately leads to their interaction with the cytoskeleton. These observations underscore the complexity of the events that are required for achieving effective interactions between MHC class II molecules and TCR, and suggest, with regard to efficient antigen presentation, that the physical state of the class II molecules is at least as important as their signal transducing capacity.
- Published
- 1994
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43. Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.
- Author
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Young RM, Arnette JK, Roess DA, and Barisas BG
- Subjects
- Animals, Cells, Cultured, Energy Transfer, Female, Kinetics, Mice, Mice, Inbred BALB C, Microscopy, Fluorescence instrumentation, Microscopy, Fluorescence methods, Photolysis, Spectrometry, Fluorescence instrumentation, Spectrometry, Fluorescence methods, Time Factors, B-Lymphocytes metabolism, Concanavalin A metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism, Models, Theoretical
- Abstract
We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.
- Published
- 1994
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44. Class I major histocompatibility complex antigens are not associated with the LH/CG receptor on ovine luteal cells.
- Author
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Kenny N, Rahman NA, Barisas BG, and Roess DA
- Subjects
- Animals, Corpus Luteum cytology, Female, Sheep, Corpus Luteum metabolism, Histocompatibility Antigens Class I metabolism, Receptors, LH metabolism
- Abstract
We have examined the rotational dynamics of the luteinizing hormone (LH) receptor on day 10 intact ovine small luteal cells and isolated plasma membranes using polarized fluorescence depletion (PFD). This technique measures rotational correlation times which are proportional to the in-membrane volume of a protein and are useful for examining changes in protein size due to receptor aggregation or protein-protein interactions. Eosin isothiocyanate (EITC)-derivatized ovine LH (EITC-oLH) bound to the LH receptor on luteal cell plasma membranes had a rotational correlation time of 20 +/- 6 microseconds, while that for EITC-human chorionic gonadotropin (EITC-hCG)-occupied LH receptors was 46 +/- 13 microseconds. Slower rotational times for EITC-oLH and EITC-hCG, 63 +/- 19 and 87 +/- 20 microseconds, respectively, were obtained on intact ovine luteal cells. These results indicate that the LH receptor exists as a larger molecular mass complex when binding hCG than oLH, a difference which could be attributable to hCG-induced LH-receptor interaction with additional membrane protein(s). One candidate protein for such an interaction is the Major Histocompatibility Complex (MHC) Class-I antigen. However, the rotational correlation time of EITC-anti-MHC Class-I antibody (SBU I) Fab fragments was 247 +/- 34 microseconds, indicating that MHC Class I is located in complexes larger than those identified by EITC-OLH or EITC-hCG. Preincubation of plasma membranes with 1 nM unlabeled oLH or hCG had no significant effect on this rotational correlation time. Further, treatment of cells with SBU I had no affect on either basal or oLH-stimulated progesterone secretion. Thus it appears that the ovine luteal LH-receptor is not associated with MHC Class I and that antibody-induced aggregation of MHC Class I does not cause an LH-mimetic response.
- Published
- 1993
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45. Membrane changes in lipopolysaccharide-stimulated murine B lymphocytes associated with cell activation.
- Author
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Printen JA, Woodard SL, Herman JR, Roess DA, and Barisas BG
- Subjects
- Animals, B-Lymphocytes drug effects, Carbocyanines chemistry, Female, Lipopolysaccharides, Membrane Fluidity, Mice, Mice, Inbred BALB C, Mice, Inbred C3H, Rhodamines, Temperature, B-Lymphocytes chemistry, Cell Membrane chemistry, Lymphocyte Activation
- Abstract
The lateral diffusion of the fluorescent lipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured in the membranes of murine B lymphocytes treated with the B cell mitogen lipopolysaccharide (LPS). The mobility of DiI, as measured by fluorescence photobleaching recovery (FPR) techniques, was temperature-dependent with a value of 6.1.10(-9) cm2 s-1 at 37 degrees C. Untreated cells exhibited this diffusion coefficient over 72 h in culture. In contrast, DiI mobility decreased to 2.0.10(-9) cm2 s-1 at 37 degrees C in membranes of LPS-stimulated lymphocytes 24 h following LPS exposure. Interestingly, this decreased lipid lateral diffusion was not accompanied by any change in surface immunoglobulin lateral diffusion which remained essentially unchanged at 3.6-4.3.10(-11) cm2 s-1 over 72 h. To determine whether LPS effects on lipid lateral diffusion were due to insertion of LPS into the cell plasma membrane, we examined TRITC-LPS diffusion in B lymphocytes from LPS-responsive Balb/c and C3Heb/FeJ mice and from hypo-responsive C3H/HeJ mice. DiI and TRITC-LPS mobility decreased more than 50% in LPS-stimulated Balb/c and C3Heb/FeJ cells by 72 h. On C3H/HeJ lymphocytes, there was no change in DiI or TRITC-LPS lateral diffusion throughout the incubation period. These data indicate that B lymphocyte membrane composition is altered in LPS-activated lymphoblasts and that the decreased lateral diffusion of lipid probes does not result from membrane perturbation by LPS insertion into the lipid bilayer. Further, similarities between TRITC-LPS and DiI lateral diffusion suggest that most LPS molecules interact non-specifically with B cell membranes, presumably by acyl chain insertion of the lipid A moiety.
- Published
- 1993
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- View/download PDF
46. Molecular dynamics of luteinizing hormone receptors on rat luteal cells.
- Author
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Roess DA, Rahman NA, Kenny N, and Barisas BG
- Subjects
- Animals, Cells, Cultured, Chorionic Gonadotropin metabolism, Corpus Luteum cytology, Diffusion, Female, Fluorescence Polarization, Fluorescent Dyes, Humans, Luteinizing Hormone metabolism, Rats, Sheep, Corpus Luteum metabolism, Receptors, LH metabolism
- Abstract
To better understand the in situ organization of the luteinizing hormone receptor on rat luteal cells, we have examined the molecular motions of this receptor following binding of ovine luteinizing hormone (oLH) or human chorionic gonadotropin (hCG). Fluorescence photobleaching recovery (FPR) measurements of LH receptor lateral diffusion were performed using tetramethylrhodamine isothiocyanate (TRITC)-derivatized oLH or hCG as a probe. These experiments indicate that TRITC-oLH occupied LH receptors on luteal cells obtained from superovulated female rats have a lateral diffusion coefficient D of (1.7 +/- 0.6).10(-10) cm2s-1 at 27 degrees C with fluorescence recovery after photobleaching of 46 +/- 5%. In similar experiments, binding of TRITC-hCG caused a significant decrease in LH receptor lateral diffusion; fluorescence recovery after photobleaching was less than 20%. To determine whether hCG-occupied receptors might exist in large aggregates, we measured the rotational correlation times (RCT) of hCG and oLH bound to the LH receptor on intact cells using single cell polarized fluorescence depletion (PFD). At 4 degrees C, LH receptors occupied by eosin isothiocyanate (EITC)-hCG exhibited a slower RCT (64 microseconds) than did receptors occupied by EITC-oLH (43 microseconds). At this temperature both TRITC-oLH and TRITC-hCG occupied LH receptors were laterally immobile. These FPR and PFD results suggest that the molecular motions of the luteal cell LH receptor are significantly modulated by the subtle structural differences in various bound gonadotropins.
- Published
- 1992
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47. Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.
- Author
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Chakrabarti A, Matko J, Rahman NA, Barisas BG, and Edidin M
- Subjects
- B-Lymphocytes immunology, Cell Line, Cell Membrane immunology, Diffusion, Erythrosine chemistry, Flow Cytometry, Fluorescence Polarization, Humans, Immunoglobulin Fab Fragments, Proteolipids, HLA-A2 Antigen immunology, Liposomes immunology
- Abstract
Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
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48. Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts.
- Author
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Boullier JA, Peacock JS, Roess DA, and Barisas BG
- Subjects
- Animals, Avian Sarcoma Viruses physiology, Carbocyanines, Cells, Cultured, Chick Embryo, Concanavalin A, Diffusion, Fibroblasts metabolism, Kinetics, Rhodamines, Temperature, Cell Transformation, Viral physiology, Membrane Glycoproteins metabolism, Membrane Lipids metabolism
- Abstract
We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.
- Published
- 1992
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49. Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion.
- Author
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Rahman NA, Pecht I, Roess DA, and Barisas BG
- Subjects
- Animals, Antibodies, Monoclonal, Biophysical Phenomena, Biophysics, Fluorescence Polarization, Immunoglobulin E, Immunoglobulin Fab Fragments, Mast Cells chemistry, Membrane Proteins chemistry, Rats, Tumor Cells, Cultured chemistry, Tumor Cells, Cultured immunology, Mast Cells immunology, Receptors, Fc chemistry
- Abstract
We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon receptors (Fc epsilon RI) on rat mucosal mast cells of the RBL-2H3 line in their resting monomeric and differently oligomerized states using as probes IgE and three monoclonal antibodies (mAbs; H10, J17, and F4) specific for the Fc epsilon RI. PFD experiments using eosin (EITC)-IgE show that individual Fc epsilon RI on cells have a rotational correlation time (RCT) at 4 degrees C of 79 +/- 4 microseconds. Similarly, Fc epsilon RI-bound EITC-Fab fragments of the J17 Fc epsilon RI-specific mAb exhibit an RCT of 76 +/- 6 microseconds. These values agree with previous measurements of Fc epsilon RI-bound IgE rotation by time-resolved phosphorescence anisotropy methods. Receptor-bound EITC-conjugated divalent J17 antibody exhibits an increased RCT of 140 +/- 6 microseconds. This is consistent with the ability of this mAb to form substantial amounts of Fc epsilon RI dimers on these cell surfaces. The ratio of limiting to initial anisotropy in these experiments remains constant at about 0.5 from 5 degrees C through 25 degrees C for IgE, Fab, and intact mAb receptor ligands. Extensive cross-linking by second antibody of cell-bound IgE, of intact Fc epsilon RI-specific mAbs or of their Fab fragments, however, produced large fixed anisotropies demonstrating, under these conditions, receptor immobilization in large aggregates. PFD using the mAbs H10 and F4 as receptor probes yielded values for triplet lifetimes, RCT values, and anisotropy parameters essentially indistinguishable from those obtained with the mAb J17 clone. Possible explanations for these observations are discussed.
- Published
- 1992
- Full Text
- View/download PDF
50. Triamcinolone acetonide inhibits lymphocyte differentiation in B cells decorated with artificial antigen receptors.
- Author
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Roess DA, Zschokke ME, Peacock JS, and Barisas BG
- Subjects
- 2,4-Dinitrophenol, Antigens, Bacterial, B-Lymphocytes cytology, B-Lymphocytes drug effects, Cell Differentiation drug effects, Cells, Cultured, Dinitrophenols pharmacology, Flagellin immunology, Flagellin pharmacology, Humans, Kinetics, Salmonella, Antibodies, Monoclonal immunology, B-Lymphocytes immunology, Triamcinolone Acetonide pharmacology
- Abstract
We examined the effects of triamcinolone acetonide (TA) on T cell independent antigen-induced differentiation of human B cells. Purified human B cells artificially decorated with palmitate-conjugated monoclonal IgA antibody specific for 2,4-dinitrophenyl differentiated polyclonally when challenged with optimum concentrations of dinitrophenyl-derivatized polymerized flagellin. This B cell response was reduced by the synthetic corticosteroid TA at a concentration of 10(-6) M. This suggests that TA can inhibit in vitro B lymphocyte differentiation independent of T cells.
- Published
- 1991
- Full Text
- View/download PDF
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