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N L Thompson

Publications and source records attributed to N L Thompson.

At least 19 recordsLinked to original sources
PubMed1992

Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.

Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Direct measurement of the weak interactions between a mouse Fc receptor (Fc gamma RII) and IgG1 in the absence and presence of hapten: a total internal reflection fluorescence microscopy study.

Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.

Animals↗
PubMed1992

Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Animals↗
PubMed1992

Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.

Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.

Animals↗
PubMed1992

Evanescent interference patterns for fluorescence microscopy.

The increasing experimental use of total internal reflection/fluorescence photobleaching recovery has motivated a theoretical study of the spatial intensity profiles generated by two interfering evanescent waves. The interference patterns generated by evanescent waves differ considerably from those generated by plane waves in a homogenous medium because evanescent waves are not transverse and because the evanescent propagation number depends on the incidence angle of the totally internally reflected light. The periodicity and contrast of the evanescent interference patterns under various conditions are calculated; these parameters depend on the intensities, polarizations, and incidence angles of the two incident beams, as well as the refractive indices of the two media that form the planar interface where total internal reflection occurs. The derived intensity profiles are used to develop expressions for the shapes of fluorescence photobleaching recovery curves when evanescent interference patterns are used for fluorescence excitation and bleaching. The calculations also suggest that colliding beam experiments may confirm theoretically predicted evanescent field polarizations.

Binding Sites↗
PubMed1992

Distribution of transforming growth factor beta in a two-week-old human embryo.

Using immunohistochemical methods we have investigated the presence of transforming growth factors beta 1, beta 2 and beta 1 precursor in a two-week-old bilaminar human embryo. TGF-beta 1 precursor was seen in both the epiblast and the hypoblast. In contrast to the widespread localization of TGF-beta 1 precursor in the embryo proper, antibodies to the mature TGF-beta 1 peptide localized preferentially to the hypoblast with only weak staining in the epiblast. Staining with antibodies to TGF-beta 2 was generally weak in both the epiblast and hypoblast layers of the embryo proper. These results show that TGF-beta 1 and beta 2 peptides are detectable as early as the second week of human development.

Embryo, Mammalian↗
PubMed1991

Evidence from total internal reflection fluorescence microscopy for calcium-independent binding of prothrombin to negatively charged planar phospholipid membranes.

Measurements to test for a proposed Ca2+-independent interaction of prothrombin with membranes containing acidic phospholipids are described. Fluorescein-labeled bovine prothrombin and its amino- and carboxy-terminal peptides, prothrombin fragment 1 and prethrombin 1, were added at various concentrations in the presence or absence of Ca2+ to the aqueous space bathing substrate-supported planar membranes composed of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), POPC/bovine brain phosphatidylserine (bovPS) (70:30 mol/mol), or POPC/1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) (70:30 mol/mol). Total internal reflection fluorescence microscopy (TIRFM) at the membrane-solution interface showed a significant enhancement by acidic lipids of prothrombin and prothrombin fragment 1 binding in the presence of 5 mM Ca2+, with apparent dissociation constants of 0.4 and 1 microM, respectively. TIRFM measurements indicated that bovPS and DOPG also significantly enhanced the binding of fluorescein-labeled prothrombin to the planar membranes in the absence of Ca2+, with apparent dissociation constants (13-30 microM) at least an order of magnitude larger than the Ca(2+)-dependent constant for prothrombin binding. Association of prethrombin 1 but not prothrombin fragment 1 with membranes in the absence of Ca2+ was enhanced by the presence of bovPS in the membranes, which suggests that the Ca(2+)-independent binding site(s) is (are) in the prethrombin 1 but not the fragment 1 portion of prothrombin.

Animals↗
PubMed1991

Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.

Animals↗
PubMed1991

Binding and mobility of anti-dinitrophenyl monoclonal antibodies on fluid-like, Langmuir-Blodgett phospholipid monolayers containing dinitrophenyl-conjugated phospholipids.

The association of a fluorescently labelled anti-dinitrophenyl monoclonal antibody (ANO2) with Langmuir-Blodgett monolayers composed of three different binary mixtures of phosphatidylcholine and dinitrophenyl-conjugated phosphatidylethanolamine has been characterized. Quantitative fluorescence microscopy measurements demonstrated that measurable amounts of antibodies bound to the monolayers only at high molar fractions of dinitrophenyl-conjugated lipid (greater than or equal to 5 mol%). Fluorescence pattern photobleaching recovery measurements showed that the apparent translational diffusion coefficients and mobile fractions of a fluorescent lipid were high for all monolayer compositions and that the antibody translational mobility was measurable but slow and depended on the two-dimensional antibody density. The results demonstrate that the ANO2-binding characteristics of Langmuir-Blodgett monolayers containing dinitrophenyl-conjugated phospholipids are substantially different from those of similar model systems but that the ANO2 antibodies, when bound, display similar diffusive behavior.

Antibodies, Monoclonal↗
PubMed1991

A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model.

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue.

Animals↗
PubMed1990

Identification and characterization of a rat hepatic oncofetal membrane glycoprotein.

A major interest of our laboratory is to delineate the pathways leading to experimentally induced liver cancer in the rat. Although the cellular progenitors of primary hepatocellular carcinoma remain controversial, current findings suggest that proliferation of chemically initiated liver epithelial cells gives rise to hepatic nodules, a rare population of which eventually progress to carcinoma. Presently, the availability of cell surface markers that are closely associated with malignant progression is needed for the identification, isolation, and further characterization of these rare malignant cells. In this paper, we describe two new monoclonal antibodies (MAbs), MAb 324.5 and MAb 324.9, that recognize a novel oncofetal membrane glycoprotein, designated TuAg1. MAbs 324.5 and 324.9 were produced using three different transplantable hepatocellular carcinoma cell lines during immunization and screening. MAb 324.5 and MAb 324.9 were shown to be reactive with different epitopes on TuAg1 by competitive immunoprecipitation assays combined with results from immunodepletion analysis and one-dimensional V-8 peptide maps. TuAg1 showed variations in molecular weight from 78,000 to 92,000, and a marked heterogeneity in pI, with charge variants ranging between 4.3 and 6.0. The 324.5-epitope was not expressed at detectable levels in any adult normal tissues or during liver regeneration but was transiently expressed during fetal liver development as shown by indirect immunofluorescence analysis of frozen tissue sections. In contrast, the 324.9-epitope was observed on nerve fibers and ganglia and on sperm tails in the adult rat and also appeared independently of the 324.5-epitope during fetal development. Although normal hepatocytes did not express TuAg1, isolated hepatocytes became positive during the first 24 h of primary culture. Attempts to modulate the in vitro expression of TuAg1 were unsuccessful; however, TuAg1 was lost within 7 days following ectopic transplantation of cultured hepatocytes into the pancreas. During the carcinogenic process, TuAg1 was expressed by a rare population of hepatic nodules, by many primary liver tumors, and by all lung metastases and transplantable hepatocellular carcinomas examined to date. Taken together, these observations suggest that the in vivo constitutive expression of this novel oncofetal membrane antigen is closely associated with acquisition of the malignant phenotype during hepatocarcinogenesis.

Animals↗
PubMed1990

Mediation of wound-related Rous sarcoma virus tumorigenesis by TGF-beta.

In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-beta is present locally shortly after wounding, but not unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor-beta 1 (TGF-beta 1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-beta resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in wound healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-alpha had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-beta release during the wound-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system.

Animals↗
PubMed1990

Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy.

A procedure for constructing substrate-supported planar membranes using membrane fragments isolated from the macrophage-related cell line J774A.1 is described. Total internal reflection (TIR) fluorescence microscopy is employed to demonstrate that fluorescently labeled Fab fragments of a monoclonal antibody (2.4G2) with specificity for a murine macrophage cell-surface receptor for IgG (moFc gamma RII) bind to the planar model membranes. These measurements show that the planar membranes contain moFc gamma RII and yield a value for the association constant of 2.4G2 Fab fragments with moFc gamma RII equal to (9.6 +/- 0.4) x 10(8) M-1 and indicate that the surface density of reconstituted moFc gamma RII is approximately 50 molecules/microns 2. In addition, TIR fluorescence microscopy is used to investigate the Fc-mediated competition of unlabeled, polyclonal murine IgG with labeled 2.4G2 Fab fragments for moFc gamma RII in the planar membranes. These measurements indicate that the reconstituted moFc gamma RII recognized by 2.4G2 Fab fragments also retains the ability to bind murine IgG Fc regions and yield a value for the association constant of polyclonal murine IgG with moFc gamma RII equal to (1-5) x 10(5) M-1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions.

Animals↗
PubMed1990

Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.

Animals↗
PubMed1990

Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy.

The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites.

Antibodies, Monoclonal↗
PubMed1990

Effects of newborn screening of cystic fibrosis on reported maternal behaviour.

Screening for cystic fibrosis is highly controversial. Concerns have been expressed that newborn screening may cause mothers, who had considered their child to be healthy before diagnosis, to overprotect their child. Some critics of screening also suggest that a period of delay from onset of symptoms to diagnosis may help a mother adjust to the reality of the child's lethal condition. This study compared the strength of overprotective child rearing attitudes of 29 mothers whose children were screened (13 had symptomatic children and 16 asymptomatic children) with the attitudes of 29 mothers whose children were diagnosed after the onset of symptoms. Results indicate that newborn screening had not increased a mother's tendency to overprotect her child with cystic fibrosis and in some cases the tendency had decreased. Further, delay in diagnosis when screening was not conducted usually caused mothers considerable personal distress.

Anxiety↗
PubMed1990

Transforming growth factor-beta expression in fibropapillomas induced by bovine papillomavirus type 1, in normal bovine skin, and in BPV-1-transformed cells.

There is substantial evidence to suggest that transforming growth factor-beta (TGF-beta) plays an important role in wound healing and tissue repair as well as in carcinogenesis. It has also been observed that naturally occurring bovine papillomavirus type 1 (BPV-1)-induced bovine fibropapillomas occur predominantly at traumatized sites of the body, suggesting that humoral factors released in wounds might be important for papillomavirus infection. We have therefore investigated the possible role of TGF-beta 1 in BPV-1 infections. Two antipeptide antibodies which recognize different epitopes in the N-terminus of TGF-beta 1 were used to localize TGF-beta 1 in bovine fibropapillomas and normal bovine skin using immunohistochemical methods. Staining by anti-LC(1-30) is intracellular in suprabasal keratinocytes of the epidermis as well as the hair follicles and sebaceous glands and correlates with known sites of TGF-beta 1 mRNA synthesis. Anti-CC(1-30) staining is extracellular in the immediately underlying dermis. Neither the pattern nor intensity of TGF-beta 1 staining was affected by BPV-1 infection. C127 cells and BPV-1-transformed C127 cells were compared for TGF-beta 1 mRNA expression and secretion of TGF-beta 1 peptide. Although the levels of messenger RNA and secreted TGF-beta 1 peptide were similar in both cell types, five- to six-fold greater amounts of TGF-beta-like activity per cell was detected in media conditioned by the uninfected cells. TGF-beta 1 treatment had no effect on the growth rate of either cell type or on BPV-1 gene expression in the transformed cells.

Animals↗