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N L Thompson

Publications and source records attributed to N L Thompson.

At least 91 records · Page 5Linked to original sources
PubMed1987

Theory of sample translation in fluorescence correlation spectroscopy.

New applications of the technique of fluorescence correlation spectroscopy (FCS) require lateral translation of the sample through a focused laser beam (Peterson, N.O., D.C. Johnson, and M.J. Schlesinger, 1986, Biophys. J., 49:817-820). Here, the effect of sample translation on the shape of the FCS autocorrelation function is examined in general. It is found that if the lateral diffusion coefficients of the fluorescent species obey certain conditions, then the FCS autocorrelation function is a simple product of one function that depends only on transport coefficients and another function that depends only on the rate constants of chemical reactions that occur in the sample. This simple form should allow manageable data analyses in new FCS experiments that involve sample translation.

Kinetics↗
PubMed1987

Role of transforming growth factor-beta in the development of the mouse embryo.

Using immunohistochemical methods, we have investigated the role of transforming growth factor-beta (TGF-beta) in the development of the mouse embryo. For detection of TGF-beta in 11-18-d-old embryos, we have used a polyclonal antibody specific for TGF-beta type 1 and the peroxidase-antiperoxidase technique. Staining of TGF-beta is closely associated with mesenchyme per se or with tissues derived from mesenchyme, such as connective tissue, cartilage, and bone. TGF-beta is conspicuous in tissues derived from neural crest mesenchyme, such as the palate, larynx, facial mesenchyme, nasal sinuses, meninges, and teeth. Staining of all of these tissues is greatest during periods of morphogenesis. In many instances, intense staining is seen in mesenchyme when critical interactions with adjacent epithelium occur, as in the development of hair follicles, teeth, and the submandibular gland. Marked staining is also seen when remodeling of mesenchyme or mesoderm occurs, as during formation of digits from limb buds, formation of the palate, and formation of the heart valves. The presence of TGF-beta is often coupled with pronounced angiogenic activity. The histochemical results are discussed in terms of the known biochemical actions of TGF-beta, especially its ability to control both synthesis and degradation of both structural and adhesion molecules of the extracellular matrix.

Animals↗
PubMed1987

Helene Deutsch: a life in theory.

Evaluations of Helene Deutsch's work on female psychology almost invariably focus on her idealization of motherhood and on her attribution of narcissism, passivity, and masochism to the "feminine" woman. The author suggests that identification plays a more important role in Deutsch's portrayal of feminine development than has hitherto been acknowledged. Deutsch treats identification as a reparative process that enables women to overcome major traumata by allowing them to re-experience the initial bliss of the mother-child relationship. The biographical origins of Deutsch's theory of feminine development are explored, and an assessment offered of how her reliance on personal experience both enhanced and inhibited its explanatory power.

Europe↗
PubMed1986

Total internal reflection fluorescence. Measurement of spatial and orientational distributions of fluorophores near planar dielectric interfaces.

The fluorescence collected from a fluorophore which is near a planar interface and is excited by a laser beam that is totally internally reflected at the interface depends on the direction of the absorption and emission transition dipole moments of the fluorophore with respect to the interface, on the distance from the fluorophore to the interface, on the angle of incidence and polarization direction of the exciting beam, and on properties of the collection optics. Expressions are derived for the excitation and subsequent emission and collection of fluorescence from a population of fluorophores near a planar interface. Presented is a general model-independent method of obtaining characteristic parameters of the spatial and orientational distribution of the population of fluorophores, from a measure of the fluorescence collected as a function of the polarization and the incidence angle of the totally internally reflected laser beam. The method is illustrated with several simulation calculations.

Lasers↗
PubMed1986

Proto-oncogene expression and growth factors during liver regeneration.

When growth is stimulated in normally quiescent hepatocytes, steady-state levels of c-fos, c-myc, and p53 mRNAs increase sequentially and transiently before DNA replication. C-fos mRNA increases almost immediately after partial hepatectomy and decreases by 2 hr; c-myc mRNA reaches maximal levels between 30 min and 2 hr. In contrast, the p53 mRNA increase corresponds to the G1/S transition, and mRNAs from c-ras genes are elevated later, coinciding with DNA replication and mitosis. p53 and p21 proteins are elevated when their mRNAs are more abundant. This regulated response suggests that these genes either control key steps in the cell cycle or are responding to humoral or internal growth factors acting at specified growth stages. We propose that hepatocytes go through a "priming" stage during the first four hours after partial hepatectomy and that their progression through late G1, is likely to be controlled by autocrine or paracrine mechanisms, which may account for the precisely regulated growth of the liver after partial hepatectomy. Transforming growth factor beta (TGF beta) is a potent inhibitor of DNA synthesis in normal hepatocytes in vitro. We show that TGF beta mRNA increases in the regenerating liver at the time of hepatocyte DNA synthesis and mitosis. In normal or regenerating liver, the mRNA for this growth factor is contained in nonparenchymal cells but not in hepatocytes. We suggest that TGF beta may be a component of a paracrine regulatory loop that controls hepatocyte replication.

Animals↗
PubMed1986

Sequential protooncogene expression during rat liver regeneration.

When growth is stimulated in the normally quiescent adult rat liver by partial hepatectomy, steady state levels of messenger RNAs (mRNAs) for c-fos, c-myc, and p53 increase sequentially during the prereplicative phase which precedes DNA synthesis. Levels of c-fos mRNA are elevated at least 4-fold within 15 min after partial hepatectomy and decrease rapidly by 2 h; c-myc mRNA reaches maximal levels (5-fold over normal) between 30 min and 2 h after the operation. A second, transient phase of expression for both c-fos and c-myc occurs around 8 h after partial hepatectomy. p53 mRNA levels increase between 8 and 12 h after the operation (5-fold over normal) and are reflected in an elevation of steady state levels of p53 protein between 12 and 15 h after partial hepatectomy. The levels of ras p21 protein increase much later at a time of active DNA replication and cell division. Actinomycin D injected at the time of partial hepatectomy blocks the increase in c-myc at 2 h but has no effect on c-fos mRNA levels. Actinomycin D injected at 6 h only partially blocks the increase in c-myc and p53 mRNA at 8 h but does not affect c-fos mRNA. Our results suggest that the transient and sequential expression of protooncogenes during the prereplicative stage of liver regeneration is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle and can serve as markers for identifying specific humoral factors involved in liver regeneration.

Albumins↗
PubMed1986

A structural analysis of gap and tight junctions in the rat liver during a dietary treatment that induces oval cell proliferation.

The authors have investigated early changes in liver cell gap and tight junctions that occur when rats are fed a carcinogenic diet. Animals were fed a choline-deficient diet that contained 0.1% ethionine (CDE) for periods up to 6 weeks. Short-term feeding of this diet results in the rapid proliferation of so-called "oval cells" within the liver, which is reversible upon returning the rats to a normal diet. Livers from animals fed the diet were removed at various times during feeding and during recovery from the diet and were analyzed by light and electron microscopy. The freeze-fracture technique was used to produce extended views of the internal structure of liver cell membranes at each stage under study. The characteristic junctional complex surrounding canalicular regions in normal liver disappears after only 2 weeks of the CDE regimen. Gap junctions were not found after 4 weeks of the diet, and tight junctions became increasingly disorganized. Tight junction elements were observed, however, between hepatocytes and oval cells, which indicated that these two cell types do interact directly. Changes occur in the structural complexity of tight junction elements between hepatocytes and between hepatocytes and oval cells. Recovery from the CDE diet results in a rapid increase in junctional complexity, and the large gap junction plaques characteristic of normal liver are visible within 2 weeks after cessation of the CDE regimen. These and other observations demonstrate that reversible alterations in hepatocyte gap and tight junctions occur as a result of administration of a diet that induces oval cell proliferation. The relationship of these changes to those that have been reported during other processes of cell proliferation are discussed.

Animals↗
PubMed1985

Motion of myosin cross-bridges in skeletal muscle fibers studied by time-resolved fluorescence anisotropy decay.

The time-resolved fluorescence polarization anisotropy signal has been measured from fluorescent-labeled myosin cross-bridges in single glycerinated muscle fibers in the relaxed and rigor states. In one experimental configuration, the polarization of the excitation light and the fiber axis are aligned, and the anisotropy is sensitive to rotational motions of the probes about axes other than the fiber axis. The rotational correlation times are approximately 1000 ns for relaxed fibers and greater than 7000 ns for rigor fibers. In another experimental configuration, the excitation light polarization is perpendicular to the fiber axis, and its propagation vector has a component parallel to the fiber axis so that the anisotropy is sensitive to probe rotational motion about different axes, including the fiber axis. In this configuration, the rotational correlation times are approximately 300 ns for both relaxed and rigor fibers. The theory of rotational diffusion in a potential described in a related paper [Burghardt, T.P. (1985) Biophys. J. (in press)] is applied to the relaxed fiber data.

Animals↗
PubMed1985

The influence of diffusion on the rate of a reversible quasi-unimolecular reaction in one, two or three dimensions.

The role of diffusion in the kinetics of a reversible quasi-unimolecular reaction is considered. Equations that couple diffusion and reversible reaction are defined. From these equations are derived expressions for the concentrations of the reacting species, as a function of time, after a perturbation from their equilibrium concentrations. These expressions demonstrate how the time-dependent approach by a concentration to its equilibrium value is determined by the binding rate of adjacent molecules, the dissociation rate, the diffusion coefficients, the distance of closest approach of the reactants, the concentrations of the reactants, and the dimensionality. The expressions are applicable to perturbation-relaxation experiments in one, two, and three dimensions. The formalism is compared with previously existing theories.

Journal Article↗
PubMed1985

Model-independent electron spin resonance for measuring order of immobile components in a biological assembly.

A model-independent description of the angular orientation distribution of elements in an ordered biological assembly is applied to the electron spin resonance (ESR) technique. As in a previous model-independent treatment of fluorescence polarization (Burghardt, T.P., 1984, Biopolymers, 23:2383-2406) the elemental order is described by an angular distribution of molecular frames with one frame fixed in each element of the assembly. The distribution is expanded in a complete orthonormal set of functions. The coefficients of the series expansion (the order parameters) describe the orientation distribution of the elements in the assembly without reference to a model and can be obtained from the observed spectrum. The method establishes the limitations of ESR in detecting order in the assembly by determining which distribution coefficients the technique can detect. A method of determining the order parameters from an ESR spectra, using a set of ESR basis spectra, is developed. We also describe a treatment that incorporates the actual line shape measured from randomly oriented, immobile elements. In this treatment, no model-dependent assumptions about the line shape are required. We have applied the model-independent analysis to ESR spectra from spin-labeled myosin cross-bridges in muscle fibers. The results contain detailed information on the spin-probe angular distribution and differ in interesting ways from previous model-dependent interpretations of the spectra.

Animals↗
PubMed1985

Oncodevelopmental expression of rat placental alkaline phosphatase. Detection in oval cells during liver carcinogenesis.

Oval cells isolated from livers of rats fed a choline-deficient diet containing 0.1% DL-ethionine (CDE) have an alkaline phosphatase (ALKP) isozyme which can be distinguished by its electrophoretic mobility from the enzyme present in parenchymal cells isolated from normal liver or livers of rats fed the CDE diet for 4 weeks. The oval cell ALKP has the same electrophoretic mobility as the enzyme from fetal rat liver and placenta. ALKPs from oval cells, parenchymal cells, and placenta all differ from the intestinal enzyme by their electrophoretic mobility, isoelectric focusing, and the patterns of amino acid inhibition of enzyme activity. Oval cells in preneoplastic livers, fetal hepatocytes, and tumor cells of a primary hepatocellular carcinoma induced by CDE feeding stained with a monoclonal antibody directed against rat placental ALKP. Hepatocytes (in normal or preneoplastic livers) and bile duct cells in normal liver did not stain with the same antibody. Placental ALKP may thus be a useful marker in tracing the origin and fate of oval cells during hepatocarcinogenesis.

Alkaline Phosphatase↗
PubMed1984

Covalent linkage of a synthetic peptide to a fluorescent phospholipid and its incorporation into supported phospholipid monolayers.

A number of fluorescent peptide-lipid conjugates have been synthesized. Peptides with ten or eleven amino acids are linked through a single lysine residue to the headgroup of phosphatidylethanolamine, fluorescently labelled on one acyl chain, using homobifunctional disuccinimidyl crosslinking reagents. Peptide-lipids can be further derivatized with the hapten dinitrophenyl. Purified peptide-lipids have been incorporated into dimyristoylphosphatidylcholine monolayers at the interface of air and phosphate-buffered saline, at concentrations of up to 11 mol%. For equal average molecular areas, monolayers containing peptide-lipids have higher surface pressures than pure lipid monolayers; for equal surface pressures, peptide-lipid monolayers have higher average molecular areas than pure lipid monolayers. When the peptide-lipid monolayers are transferred to hydrophobic glass slides, the fluorescence appears uniformly distributed. Fluorescence recovery after photobleaching measurements indicate that peptide-lipids diffuse in the monolayer with coefficient 1.5 X 10(-9) cm2/s, which is much smaller than that of typical lipids in fluid membranes. In addition, the diffusion coefficient of peptide-lipids decreases with increasing peptide-lipid concentration. We conclude that the peptide portion of the peptide-lipid associates with the lipid monolayer and/or that peptide-lipids oligomerize.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection.

We consider the effect of planar dielectric interfaces (e.g., solid/liquid) on the fluorescence emission of nearby probes. First, we derive an integral expression for the electric field radiated by an oscillating electric dipole when it is close to a dielectric interface. The electric field depends on the refractive indices of the interface, the orientation of the dipole, the distance from the dipole to the interface, and the position of observation. We numerically calculate the electric field intensity for a dipole on an interface, as a function of observation position. These results are applicable to fluorescent molecules excited by the evanescent field of a totally internally reflected laser beam and thus very close to a solid/liquid interface. Next, we derive an integral expression for the electric field radiated when a second dielectric interface is also close to the fluorescent molecule. We numerically calculate this intensity as observed through the second interface. These results are useful when the fluorescence is collected by a high-aperture microscope objective. Finally, we define and calculate a "dichroic factor," which describes the efficiency of collection, in the two-interface system, of polarized fluorescence. The limit when the first interface is removed is applicable for any high-aperture collection of polarized or unpolarized fluorescence. The limit when the second interface is removed has application in the collection of fluorescence with any aperture from molecules close to a dielectric interface. The results of this paper are required for the interpretation of order parameter measurements on fluorescent probes in supported phospholipid monolayers (Thompson, N.L., H. M. McConnell, and T. P. Burghardt, 1984, Biophys. J., 46:739-747).

Electrochemistry↗
PubMed1984

Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination.

A technique is described and demonstrated for measuring the orientation distribution of fluorescent molecules in a two-dimensional system. A laser beam is totally internally reflected at the interface between a glass slide and an aqueous solution, which creates a thin layer of evanescent illumination that excites fluorescent molecules near the interface. Molecules with absorption dipoles at different tilts from the normal to the interface are preferentially excited when the laser polarization is rotated. Approximately one-half of the emitted fluorescence is collected with an inverted microscope using a high-aperture objective. The fluorescence vs. polarization curve yields the value of an order parameter that is related to the orientation distribution of absorption dipoles. This technique is applied to phospholipid monolayers made at an air/water interface and transferred to hydrophobic glass microscope slides. Dipalmitoylphosphatidylcholine monolayers were doped with 2 mol% phosphatidylethanolamine labeled with the fluorescent moiety nitrobenzoxadiazole, either on an acyl chain or on the head group. The measured value of the order parameter for the head-labeled probe decreases as a function of the surface pressure at which the monolayer is transferred to the slide, as the surface pressure increases from 10 to 40 dyne/cm. The measured value of the order parameter for the chain-labeled probe is high for all coating pressures. These results can be interpreted in terms of probe partitioning into coexistent fluid and solid domains. Dimyristoylphosphatidylcholine monolayers were doped with 2 mol% chain-labeled phosphatidylethanolamine, either free or covalently conjugated to a small peptide. In these monolayers, the measured value of the order parameter is high at all pressures. The technique presented here may also prove useful for measuring the orientation distribution of proteins bound to or embedded in a planar model membrane.

Lasers↗
PubMed1983

Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy.

An experimental application of total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) is presented. TIR/FCS is a new technique for measuring the binding and unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules in equilibrium at a surface. A laser beam totally internally reflects at the solid-liquid interface, selectively exciting surface-adsorbed molecules. Fluorescence collected by a microscope from a small, well-defined surface area approximately 5 micron2 spontaneously fluctuates as solute molecules randomly bind to, unbind from, and/or diffuse along the surface in chemical equilibrium. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the illuminated and observed region. The normalized amplitude of the autocorrelation function depends on the average number of molecules bound within the observed area. TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Using TIR/FCS, we determine that rhodamine-labeled immunoglobulin and insulin each nonspecifically adsorb to serum albumin-coated fused silica with both reversible and irreversible components. The characteristic time of the most rapidly reversible component measured is approximately 5 ms and is limited by the rate of bulk diffusion. Rhodamine-labeled bivalent antibodies to dinitrophenyl (DNP) bind to DNP-coated fused silica virtually irreversibly. Univalent Fab fragments of these same antibodies appear to specifically bind to DNP-coated fused silica, accompanied by a large amount of nonspecific binding. TIR/FCS is shown to be a feasible technique for measuring absorption/desorption kinetic rates at equilibrium. In suitable systems where nonspecific binding is low, TIR/FCS should prove useful for measuring specific solute-surface kinetic rates.

Animals↗
PubMed1983

Total internal reflection fluorescent microscopy.

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

Animals↗