Biomedical subjects

H E Gaub

Publications and source records attributed to H E Gaub.

42 records · Page 3Linked to original sources

PubMed1990

Two-dimensional recognition pattern of lipid-anchored Fab' fragments.

A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.

Antibodies, Monoclonal↗

PubMed1990

Imaging single-stranded DNA, antigen-antibody reaction and polymerized Langmuir-Blodgett films with an atomic force microscope.

The combination of an (AFM) atomic force microscope together with microfabricated cantilevers that have integrated tips opens many possibilities for imaging systems of great importance in biology. We have imaged single-stranded 25mer DNA that was adsorbed on treated mica or that was covalently bound with a crosslinker to a polymerized Langmuir-Blodgett (LB) film, the top monolayer of a bilayer system. At low magnification the AFM shows cracks between solid domains, like in an image taken with a fluorescence microscope. At higher magnification, however, the AFM reveals much finer cracks and at still higher magnification it reveals rows of individual molecules in the polymerized LB film with a spacing of 0.45 nm. We have also imaged a LB film consisting of lipids in which 4% of the lipids had hapten molecules chemically bound to the lipid headgroups. Specific antibodies can then bind to these hapten molecules and be imaged with the AFM. This points to the possibility of using the AFM to monitor selective antibody binding.

Antibodies↗

PubMed1988

Hydrodynamic hyperpolarization of endothelial cells.

The orientation and morphology of the endothelium lining the cardiovascular system may result from hemodynamic forces acting on the endothelial cells. To investigate the flow effects at the membrane level, we have examined the variations of the fluorescence intensity of two membrane-sensitive dyes, merocyanine 540 and bis(1,3-diethylthiobarbiturate)trimethineoxonol, (i) as a function of flow shear stress and (ii) with the onset or cessation of the flow. We found a time-dependent decrease in fluorescence intensity with the onset of the flow with an exponential approach to steady state of the order of 1 min. The process is reversible; when the flow is stopped the fluorescence intensity returns to its original value. The polarization of the endothelial cell membranes or, more precisely, the amplitude of the fluorescence intensity responses is an increasing function of the shear stress (up to 120 dynes/cm2). Assuming the equilibrium potential for K+ is more hyperpolarized than the resting potential and using valinomycin, we have deduced from the sign of the ionophore effects that the flow hyperpolarizes the endothelial cell membrane.

Animals↗

PubMed1986

Topological and modulated distribution of surface markers on endothelial cells.

The mobility and distribution of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) and a specific endothelial cell surface protein was assessed by fluorescein-conjugated monoclonal antibodies on bovine and murine endothelial cells grown on their extracellular matrix. The combination of data obtained from fluorescence recovery after photobleaching measurements and observations under epifluorescence and total internal reflection fluorescence reveals a restriction of these protein markers to the apical membrane of endothelial cell. This asymmetry is evident both when cells are grown at a sparse density or at confluence. When cells are brought into suspension, the fluorescein-conjugated antibody is found over the entire cell surface. The fluorescence disappears from the basal part of the cell when the cells are again spread on coverslips coated with a layer of extracellular matrix. Conversely, cells spread on glass coverslips without extracellular matrix do not show this restriction phenomenon. It is suggested that the extracellular matrix provides the signal to induce the restricted topology of membrane protein markers on endothelial cells.

Animals↗

PubMedDate not supplied

A miniaturized micro-fluorescence film balance for protein-containing lipid monolayers spread from a vesicle suspension.

In order to study protein-lipid monolayers at the air/water interface a miniaturized micro-fluorescence film-balance apparatus has been developed and combined with a modified technique of spreading and separating a monolayer from a vesicle suspension. The spreading method provides non-denaturing conditions for protein-lipids. When applied to protein-lipid vesicles, monolayers with incorporated proteins are obtained, and their thermodynamic parameters may be controlled in a well-defined way by film balance techniques. In the apparatus introduced, a movable microscope allows the observation of micro-fluorescence during the tracking of individual domains at the air/water interface of a fixed Langmuir trough. After the control of parameters such as subphase temperature, surface pressure and lateral molecule distribution, a monolayer may be transferred and immobilized on a planar solid support, making it accessible to optical surface-sensitive measuring methods as well as to electron microscopy and scanning probe techniques.

Lipids↗

PubMedDate not supplied

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field.

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.

Animals↗