Biomedical subjects

H Sigrist

Publications and source records attributed to H Sigrist.

At least 19 recordsLinked to original sources

PubMed2004

Glycoprofiling with micro-arrays of glycoconjugates and lectins.

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.

Caco-2 Cells↗

PubMed2001

Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection.

Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described.

Dimethylpolysiloxanes↗

PubMed2001

Immobilisation on polystyrene of diazirine derivatives of mono- and disaccharides: biological activities of modified surfaces.

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.

Animals↗

PubMed1998

Covalent photolinker-mediated immobilization of an intermediate dextran layer to polymer-coated surfaces for biosensing applications.

A new method is presented for the covalent binding of dextran as an intermediate layer on surface acoustic wave (SAW) devices. For biosensing applications in aqueous media commercially available SAW devices require surface passivation to prevent corrosion of the aluminum device structures in electrolytes. Thin films of polyimide and parylene revealed exceptional passivation properties. They were used as a base for dextran immobilization. Covalent binding of dextran to polymer-coated surfaces was achieved by photoimmobilization. Aryldiazirine-functionalized bovine serum albumin served as the multifunctional light-activable linking agent (photolinker polymer). Dextran and photolinker polymer were mixed and photobonded to sensor surfaces. Essential photoimmobilization parameters were optimized. The binding of proteins to dextran applying carbodiimide chemistries was exemplified with antiurease antibodies and the feasibility of specific immunosensing was investigated on SAW sensors connected to a fluid handling system.

Acoustics↗

PubMed1998

Three-dimensional extracellular matrix engineering in the nervous system.

Growing neurites are guided through their environment during development and regeneration via different cellular and extracellular matrix (ECM) molecular cues. To mimic cell-matrix interactions, a three-dimensional (3D) hydrogel-based ECM equivalent containing a covalently immobilized laminin oligopeptide sequence was designed to facilitate nerve regeneration. This study illustrates that the oligopeptide domain CDPGYIGSR covalently linked to an agarose gel as a bioartificial 3D substrate successfully supports neurite outgrowth from dorsal root ganglia (DRG) in vitro. The specificity of the neurite promoting activity was illustrated through the inhibition of neurite outgrowth from DRG in a CDPGYIGSR-derivatized gel in the presence of solubilized CDPGYIGSR peptide. Gels derivatized with CDPGYIGSK and CDPGRGSYI peptides stimulated a smaller increase of neurite outgrowth. In vivo experiments revealed the capability of a CDPGYIGSR-derivatized gel to enhance nerve regeneration in a transected rat dorsal root model compared to an underivatized gel, a CDPGRGSYI gel, and saline-filled nerve guidance channels. These data suggest the feasibility of a 3D hydrogel-based ECM equivalent capable of enhancing neurite outgrowth in vitro and in vivo.

Animals↗

PubMed1997

Immobilization of the cell-adhesive peptide Arg-Gly-Asp-Cys (RGDC) on titanium surfaces by covalent chemical attachment.

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.

Journal Article↗

PubMed1995

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde concentration in the outer monolayer.

Acylation↗

PubMed1995

Immunosensing with photo-immobilized immunoreagents on planar optical wave guides.

Immunocomplexation at wave guiding TiO2/SiO2 surfaces was investigated using an integrated optical grating coupler. For extended application of this label-free monitoring system, F(ab')2 fragments of monoclonal antibodies were photo-immobilized by photolinker polymer-mediated procedures that do not require functionalization of either the immunoreagent or the TiO2/SiO2 surface. Covalent, light-dependent binding of photolinker polymer and F(ab')2 fragments was achieved using a single-step photo-reaction. Bovine serum albumin derivatized with aryldiazirines (T-BSA) served as a photolinker polymer. T-BSA suppressed the non-specific adsorption of analytes to wave guide surfaces. Immunoreagent binding and immunological activity were analyzed and modified surfaces were investigated by scanning force microscopy. Apparent immunoreagent surface densities were 16.7 fmol F(ab')2 per mm2 sensor surface. Optical analyses revealed linear, dose-dependent antigen binding with label free analytes. Immunocompetent surfaces were regenerable by treatment at pH 2.3, rendering the immunosensing system applicable for repetitive use.

Antigen-Antibody Reactions↗

PubMed1995

Photochemical linkage of antibodies to silicon chips.

Antibodies and antigen binding fragments thereof were photochemically immobilized on surface-modified silicon chips of 5 x 5 mm size. Silicon surface-grafted diazirines and benzophenones formed covalent bonds with the immunoreagents on light activation. Photolithographic immobilization of monoclonal antibodies in aqueous media was achieved on silicon chips by activating surface-grafted benzophenones. The presence of bovine serum albumin during irradiation reduced nonspecific adsorption of the immunoreagents and retained the immunoactivity of the photoimmobilized molecules.

Animals↗

PubMed1994

Photolinker-polymer-mediated immobilization of monoclonal antibodies, F(ab')2 and F(ab') fragments.

Photolinker-polymer-mediated covalent immobilization of antibodies, F(ab') and F(ab')2 fragments has been achieved by light-dependent coupling procedures. Anti-alpha-foetoprotein (anti-AFP) monoclonal antibodies were covalently linked to microplates by layer-coating procedures, which entail antibody photoimmobilization to a photolinker-polymer-precoated surface. In this and the co-coating procedure described, diazirine-functionalized BSA (T-BSA) served as the multifunctional light-activatable linking agent (photolinker polymer). Prior to photo-activation, F(ab')2 or F(ab') fragments derived from anti-(prostate-specific antigen) monoclonal antibodies were mixed and co-coated with the photolinker polymer on to polystyrene microplates. The immunoreagents remained immunologically active after 350 nm irradiation (irradiance 0.7 mW.cm-2 for 20 min). Immuno-responses of photoimmobilized monoclonal anti-AFP antibodies were equivalent to signal intensities obtained with physically adsorbed antibodies. Photoimmobilization of anti-PSA F(ab') fragments in the presence of T-BSA revealed exponential binding characteristics indicating stabilizing molecular co-operativity of the BSA constituent. Co-coating procedures yielded 62 and 65% binding of applied 14C-labelled F(ab')2 and F(ab') fragments respectively. Covalency of antibody binding was inferred from: (i) the strict dependence of photoreagent availability; (ii) the light-dependence of the immobilization process; and (iii) the reversibility of immunocomplexation after acid treatment.

Adsorption↗

PubMed1993

Aspartic acid-212 of bacteriorhodopsin is ionized in the M and N photocycle intermediates: an FTIR study on specifically 13C-labeled reconstituted purple membranes.

Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.

Aspartic Acid↗

PubMed1992

Participation of bacteriorhodopsin active-site lysine backbone in vibrations associated with retinal photochemistry.

Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its alpha carbon (C alpha--H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the C alpha--H. The vibrational modes that exhibit this coupling are principally associated with C15--H and N--H vibrations. [C alpha--2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the C alpha--2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15--H out-of-plane wag is interpreted as a possible indication of a twist around the C15 = N bond.

Bacteriorhodopsins↗

PubMed1992

Light-dependent, covalent immobilization of biomolecules on 'inert' surfaces.

We describe a novel, versatile procedure for the light-dependent immobilization of ligands to 'inert' material surfaces. Covalent immobilization of ligands differing in chemical nature and complexity is accomplished under mild and non-destructive conditions. Topical interaction of ligands with organic or inorganic surfaces is mediated by photoactivable polymers with carbene generating trifluoromethyl-aryl-diazirines which serve as linker molecules. Light activation of aryl-diazirino functions at 350 nm yields highly reactive carbenes, and covalent coupling is achieved by simultaneous carbene insertion into both the ligand and inert surface. Thus, reactive functional groups are not required on either the ligand or the supporting material. These procedures are applicable whenever ligands, from molecules to cells--synthetically or genetically produced, or isolated from biological sources--need to be immobilized for improved performance.

Alkaline Phosphatase↗

PubMed1991

Planar bilayer membranes from photoactivable phospholipids.

Planar bilayer membranes formed from photoactivable phospholipids have been characterized by low frequency voltametry. Cyclic voltametric measurements were applied for simultaneous registration of planar membrane conductivity and capacitance. The procedure has been utilized to characterize the formation and stability of planar bilayer membranes. Bilayer membranes were formed from N'-(1,2-dimyristoyl-sn-glycero-3-phosphoethyl)-N-((m-3- trifluoromethyldiazirine)phenyl)thiourea (C14-PED), a head-group photosensitive phospholipid. In situ photoactivation of C14-PED at wavelengths greater than or equal to 320 nm altered neither the mean conductivity nor the capacitance of the bilayer. Ionophore (valinomycin) and ion channel (gramicidin) activities were not impaired upon photoactivation. In contrast, bilayer membranes formed from 1,2-bis(hexadeca-2,4-dienoyl)-sn- glycero-3-phosphocholine (C16-DENPC) revealed short life times. In situ photopolymerization of the diene fatty acids significantly increased the membrane conductivity or led to membrane rupture.

Azirines↗

PubMed1990

MP38 contains the membrane-embedded domain of the lens fiber gap junction protein MP70.

A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.

Amino Acid Sequence↗

PubMed1990

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent.

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [125I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes from Halobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[14C]-cyanate binding to BR. [125I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generated in situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1990

Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments.

For protein engineering purposes, transmembrane segments of the structurally stable protein bacteriorhodopsin have been isolated and chemically characterized. Bacteriorhodopsin was cleaved by protease V8 from Staphylococcus aureus to two fragments, V-1 and V-2. The V-2 fragment was separated by gel filtration in organic solvents and purified by reversed-phase FPLC. The fragment has been identified as the C-terminal, partially truncated double-loop of bacteriorhodopsin, including amino acids Val-167-Glu-232/4. Cleavage of V-2 by cyanogen bromide at the single Met-209 yielded two subfragments, which were purified to homogeneity by FPLC procedures. The N-terminal subfragment psi, consisted of a single transmembrane segment (helix F) of bacteriorhodopsin (Val-167-Met(Hse)-209). The C-terminal amphipathic subfragment omega, (Val-210-Glu-232/4) was identified as part of the C-terminal seventh helix of bacteriorhodopsin. Secondary structures of V-2, psi, and omega were investigated in organic solvents and micellar solutions. Native helical structures were partially retained in the solvent systems mentioned.

Amino Acid Sequence↗

PubMed1989

Heavy metal derivatives of membrane proteins: selective mercurilation of bacteriorhodopsin.

A method is described for the selective introduction of heavy atoms into structured membrane proteins by a two step modification. The procedure is applied for the purple membrane protein bacteriorhodopsin. Selective heavy-atom modification of this protein is achieved by placing a mercury reagent of intermediate polarity into phenylthiocarbamoylated bacteriorhodopsin. Incorporation of mercury requires the selective phenylthiocarbamoylation of a lysine residue. Optical investigations including circular dichroism document unchanged chromophore-protein and protein-protein interactions in mercury labeled purple membranes.

Bacteriorhodopsins↗