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B G Barisas

Publications and source records attributed to B G Barisas.

At least 55 records · Page 3Linked to original sources
PubMed1988

Glucocorticoid effects on lipid lateral diffusion and membrane composition in lipopolysaccharide-activated B-cell leukemia 1 cells.

We have investigated the effects of glucocorticoid treatment on lipid lateral diffusion and cholesterol:phospholipid ratios in the plasma membranes of the lipopolysaccharide (LPS)-responsive murine B-cell leukemia (BCL1). Exposure to LPS for 24 h at concentrations of 50 micrograms/ml, 100 micrograms/ml, and 250 micrograms/ml caused a 42% reduction in the lateral diffusion of the lipid probe 3,3'-dioctadecylindocarbocyanine iodide as measured by fluorescence photobleaching recovery techniques at 37 degrees C. In cells incubated with 50 micrograms/ml LPS, 3,3'-dioctadecylindocarbocyanine iodide diffusion is reduced to 2.2 x 10(-9) cm2s-1 compared to freshly isolated BCL1 cells where 3,3'-dioctadecylindocarbocyanine iodide diffused at a rate of 3.8 x 10(-9) cm2s-1. In BCL1 cells activated by LPS for 24 h and recultured with a pharmacological concentration of the synthetic glucocorticoid triamcinolone acetonide (TA, 10(-6) M) for 6 h, lipid lateral diffusion increased to 3.5 x 10(-9) cm2s-1. Concentrations of TA lower than 10(-6) M had no effect on lipid lateral diffusion. Six-h treatment with 10(-6) M TA had no effect on freshly isolated BCL1 cells (Time 0). However, lipid lateral diffusion increased in cells incubated 24, 48, and 72 h with LPS, and an additional 6 h with 10(-5) M TA suggested that activated BCL1 cells were more glucocorticoid sensitive than cells at Time 0. Plasma membrane cholesterol and phospholipid content, analyzed at 0 and 24 h, indicated that LPS activation was associated with a 7% increase in the cholesterol:phospholipid ratio in BCL1 membranes and that glucocorticoid treatment of these LPS-activated cells for 6 h decreased the membrane cholesterol:phospholipid ratio perhaps through inhibition of de novo cholesterol synthesis.

Animals↗
PubMed1988

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Mathematics↗
PubMed1988

Cytochalasins and colchicine increase the lateral mobility of human chorionic gonadotropin-occupied luteinizing hormone receptors on ovine luteal cells.

To determine whether cytoskeletal proteins restrict the mobility of LH-receptor complexes occupied by hCG, we measured the rate of lateral diffusion and the fraction of mobile receptor-hormone complexes by fluorescence photobleaching recovery on luteal cells treated with microtubule and microfilament disruptors and on portions of the plasma membrane separated from the underlying cytoskeleton (blebs). Enzymatically dispersed ovine luteal cells obtained on day 11 from superovulated ewes were pretreated for 1 h at 37 C with cytochalasin B (20 micrograms/ml), cytochalasin D (20 micrograms/ml), or colchicine (40 micrograms/ml). These drugs had no effect on the lateral diffusion of cell membrane glycoproteins labeled with tetramethylrhodamine isothiocyanate-derivatized succinyl Concanavalin-A. However, drug treatment caused a significant increase in the diffusion coefficient and mobile fraction of hCG-occupied LH receptors compared to values obtained on untreated cells. hCG-occupied LH receptors on untreated cells exhibited a mobile fraction of 10.3%, and these mobile complexes had a diffusion coefficient of 3.3 X 10(-11) cm2 sec-1.hCG-occupied LH receptors on cytochalasin B-treated cells had a diffusion coefficient of 9.0 X 10(-11) cm2 sec-1, with 26% fluorescence recovery. Cytochalasin D and colchicine caused significantly greater increases in hCG mobility to 22.0 and 22.5 X 10(-11) cm2 sec-1, respectively, although the mobile fraction remained at about 25-30%. That binding of hCG to the LH receptor restricts lateral mobility through interactions of the hormone-occupied receptor complex with the cytoskeleton is further indicated by results obtained on membrane blebs. The fraction of mobile hCG-occupied LH receptors on blebs was 52.3%, and complexes had a diffusion coefficient of 1.7 X 10(-10) cm2 sec-1. The hormone-induced restriction of LH receptor lateral diffusion had no effect on the lateral mobility of tetramethylrhodamine isothiocyanate-epidermal growth factor-labeled receptors.

Animals↗
PubMed1986

Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.

We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1.2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes.

In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.

Animals↗
PubMed1986

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes.

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.

Anemia, Sickle Cell↗
PubMed1986

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.

Mathematics↗
PubMed1986

Lateral diffusion of ganglioside GM1 in phospholipid bilayer membranes.

The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine. Above the transition temperature the presence of 10 mol % ganglioside GMI induced a large decrease in the rate of excimer formation. These results also indicated that the addition of ganglioside GMI to phospholipid bilayer vesicles induces a significant restriction in the lateral mobility parameters of the lipid bilayer and that the presence of Ca2' does not have a further effect in the mobility of the probe molecules.

Animals↗
PubMed1985

Glucocorticoid effects on membrane lipid mobility during differentiation of murine B lymphocytes.

The lateral motion of membrane lipids on lipopolysaccharide-stimulated murine B lymphocytes was measured using photobleaching recovery techniques. The mobility of the phospholipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured at 37 degrees C on B lymphocytes 48 h after stimulation by various concentrations of lipopolysaccharide. DiI mobility on lymphoblasts from cultures stimulated with 10 micrograms/ml lipopolysaccharide was reduced 50% compared with unstimulated, small B cells. However, both lower and higher lipopolysaccharide concentrations caused some decrease in lipid mobility. Lipid mobility was measured on B cells stimulated with 10 micrograms/ml lipopolysaccharide at zero time, on lymphoblasts at 18, 24, 48 and 72 h, and on immunoglobulin (Ig) -secreting lymphocytes at 96 h. The diffusion coefficient of DiI on both control and lipopolysaccharide-treated cells at zero time is 6.3 X 10(-9) cm2 X s-1. This value remains unchanged for unstimulated cells over 72 h. Lipid mobility of lipopolysaccharide-activated lymphoblasts decreased during incubation with lipopolysaccharide to 5.0, 3.4, 2.8 and 2.4 X 10(-9) cm2 X s-1 after 18, 24, 48 and 72 h, respectively. DiI mobility on immunoglobulin (Ig) -secreting lymphocytes identified at the foci of Protein A-coated sheep red blood cells plaques is 8.6 X 10(-9) cm2 X s-1, a value similar to that of unstimulated B cells. The effect of introducing various concentrations of a synthetic glucocorticoid, triamcinolone acetonide (TA), to 48 h lipopolysaccharide-stimulated cells for 6 h was examined. Maximal TA effect was observed at a concentration of 10(-7) M, which caused an increase in lipid mobility to 7.5 X 10(-9) cm2 X s-1. Exposing resting B cells (t = 0) or lymphoblasts (t = 24, 48 or 72 h) to TA for 3 h had no effect on lipid mobility. Treatment for 6 h with 10(-7) MTA increased DiI diffusion to 12.6, 9.9, 7.5 and 6.8 X 10(-9) cm2 X s-1 on control cells and on 24, 48 and 72 h lipopolysaccharide-activated lymphoblasts, respectively. A longer incubation of 12 h with 10(-7) MTA caused no further change in lipid lateral diffusion. The response was glucocorticoid-specific. In lymphoblasts (48 h) incubated an additional 6 h with 10(-7) MTA and a 100-fold excess of cortexolone or progesterone, the increase in lipid mobility was substantively blocked; estradiol and testosterone had no effect on lipid lateral diffusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

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.

Receptors for LH are internalized by ovine luteal cells 50 times slower when occupied by hCG than when occupied by ovine LH (oLH). To determine if differences in the rate of internalization were due to differences in the lateral mobility of the hormone-receptor complexes in the cell membrane, the diffusion coefficients of oLH- and hCG-LH receptor complexes were measured using fluorescence photobleaching recovery methods. Tetramethylrhodamine isothiocyanate (TRITC)-labeled oLH and hCG, which retained full ability to bind to receptor, were bound to LH receptors on enzymatically dispersed ovine luteal cells. Molecules labeled with TRITC within a 3-micron 2 region of the cell surface were bleached by a 500-msec pulse of 3 mW laser light at a wavelength of 514.5 nm. The laser beam intensity was then attenuated 20,000-fold, and fluorescence from the bleached area was measured by single photon counting as unbleached fluorescent hormone-receptor complexes diffused into the region. Data were analyzed on-line by a NOVA 3/12 computer. The oLH-LH receptor complex had a diffusion coefficient of 1.9 +/- 1.0 X 10(-10) cm2/sec-1, a value comparable to that of cell surface proteins nonspecifically labeled with succinylated Concanavalin A. Fluorescence recovery after photobleaching was 35%. In contrast, hCG-LH receptor complexes were immobile on the time scale of the experiment, implying that the diffusion coefficient was substantially less than 1 X 10(-11) cm2/sec-1. Deglycosylated hCG-TRITC bound to LH receptor had a diffusion coefficient (1.1 +/- 0.1 X 10(-10) cm2/sec-1) similar to that of receptors occupied by oLH. Thus, it appears that the carbohydrate portion of the hCG molecule plays a role in decreasing the mobility of the receptor for LH. These data demonstrate that the rate of lateral movement of the LH receptor in the plasma membrane of luteal cells appears to be modulated by the nature of the bound hormone.

Animals↗
PubMed1984

Thermodynamic linkages in rabbit muscle pyruvate kinase: analysis of experimental data by a two-state model.

A concerted, allosteric model is developed, and equations are derived for quantitative interpretation of the kinetic and equilibrium binding data of rabbit muscle pyruvate kinase at pH 7.5 and 23 degrees C. The simplest model which seems likely to rationalize the experimental data involves two conformational states. In this model, two simplifying assumptions are made. First, the affinities of pyruvate kinase for both substrate and inhibitor are assumed to depend only upon the conformational state of the tetrameric enzyme. Second, the rate of product formation is also assumed to depend only upon the enzyme conformation. All types of experimental data are analyzed simultaneously to estimate the parameters which best predict the total body of experimental results. The fitted parameters indicate that the intrinsic allosteric equilibrium favors the active (R) state by 11 to 1. The substrate phosphoenolpyruvate binds preferentially, by a factor of 10, to the R state, whereas the inhibitor Phe has about 23 times higher affinity for the inactive (T) state. In all cases tested, the calculated values are in good agreement with the experimental data.

Allosteric Site↗
PubMed1984

Herpes simplex virus binding and entry modulate cell surface protein mobility.

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.

Animals↗
PubMed1983

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide.

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.

Animals↗
PubMed1983

Photobleaching recovery studies of T-independent antigen mobility on antibody-bearing liposomes.

A physicochemical model for the antigen-specific B cell membrane was prepared by incorporating palmitate-conjugated MOPC 315, an anti-DNP IgA, into 8-micron diameter liposomes prepared from phosphatidylcholine, cholesterol, and cardiolipin (2:2:1). With this model system, we examined how the purely passive cross-linking of membrane-bound immunoglobulin molecules by antigen affects the lateral mobility of antigen-receptor complexes. At 37 degrees C, liposome-bound tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-DNP antibody diffuses at 1.4 X 10(-8) cm2 sec-1, a rate comparable to that observed for the phospholipid analog dil-C18-(3). Both substances exhibit complete fluorescence recovery after bleaching. The binding of TRITC-conjugates of the antigens DNP-polymerized flagellin (DNP-POL) and DNP-dextran (DNP-DEX) to liposomes bearing nonfluorescent palmitoyl-MOPC 315 was then examined. The diffusion coefficients D observed for bound antigens decrease monotonically with increased antigen dose and epitope density. For low epitope density antigens, the DNP-DEX and DNP-POL complexes are almost completely mobile. At higher epitope densities, a fraction of bound antigen appears immobile on the time scale of the experiment. This fraction is dependent on antigen concentration and epitope density and on the amount of palmitoyl MOPC 315 incorporated. The immobile fraction is 31.5% for DNP4.0-POL at 30 micrograms/ml on liposomes bearing 150,000 immunoglobulin molecules. Under these conditions D for mobile antigen is 5.0 X 10(-10) cm2 sec-1. The observed immobile fractions may represent formation of a two-dimensional gel phase of antigen-immunoglobulin aggregates. The results obtained in this study are compared with those obtained in previous work on antigen-specific mouse B lymphocytes.

Animals↗
PubMed1981

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery.

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 X 10(-11) cm2 sec-1 for DNP0.4-POL at 0.15 microgram/ml to 0.8 X 10(-11) cm2 sec-1 for DNP3.5-POL at 30 microgram/ml. For receptor bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 X10(-11) cm2 sec-1 observed for single sIg receptors. By an epitope density of 4.8, D has fallen to 2.1 X 10(-11) cm2 sec-1. Peak immunogenicities for DNP-POL and DNP-DEX are observed when antigen-receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single sIg molecule.

Animals↗
PubMed1981

Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin.

We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.

Animals↗