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Biomedical subjects

D W Grainger

Publications and source records attributed to D W Grainger.

32 records · Page 2Linked to original sources
PubMed1995

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

Animals↗
PubMed1993

Phase separated anionic domains in ternary mixed lipid monolayers at the air-water interface.

A series of ternary mixed monolayers containing varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and equimolar additions of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LYSO-PC) and palmitic acid (PA) were studied at the air-water interface. These mixed monolayers were used to model phospholipid biomembrane interfaces resulting from phospholipase A2 (PLA2) hydrolysis. Recent work [D.W. Grainger A. Reichert, H. Ringsdorf and C. Salesse (1989) Biochim. Biophys. Acta. 1023, 365-379] has shown that PLA2 hydrolysis of pure phospholipid monolayers results in formation of large PLA2 domains at the air-water interface. These domains are proposed to result from PLA2 adsorption to phase separated regions in the hydrolyzed monolayer. To elucidate the phase behaviour in these monolayer systems, surface pressure-area isotherms were measured for the ternary mixtures on pure water and buffered subphases. Fluorescence microscopy at the air-water interface was used to image fluorescent probe-doped monolayer mixtures during isothermal compressions. A water-soluble cationic carbocyanine dye was used to probe the interfacial properties of the mixed monolayers. Isotherm data do not provide unambiguous evidence for either phase separation or ideal mixing of monolayer components. Fluorescence microscopy is more revealing, showing that lateral phase separation of microstructures containing palmitic acid occurred only when monolayer subphases contained Ca2+ ions at alkaline pH. At either low pH or on Ca(2+)-free subphases, phase separation was not observed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.

Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interestingly, the observed degrees of quenching were nearly independent of the lipid membrane model studied, but directly correlated with the chemical structure of the lipids. In all cases, the antibody recognized and quenched most efficiently a lipid based on dioctadecylamine where fluorescein is attached to the headgroup via a long, flexible hydrophilic spacer. Dipalmitoyl phosphatidylethanolamine containing a fluorescein headgroup demonstrated only partial binding/quenching. Egg phosphatidylethanolamine with a fluorescein headgroup showed no susceptibility to antibody recognition, binding, or quenching. Formation of two-dimensional protein domains upon antibody binding to the fluorescein-lipids in monolayers is also presented. Chemical and physical requirements for these antibody-hapten complexes at membrane surfaces have been discussed in terms of molecular dynamics simulations based on recent crystallographic models for this antibody-hapten complex (Herron et al., 1989. Proteins Struct. Funct. Genet. 5:271-280).

Animals↗
PubMed1992

Surface characterization of 2-hydroxyethyl methacrylate/styrene copolymers by angle-dependent X-ray photoelectron spectroscopy and static secondary ion mass spectrometry.

The surface composition and structure of three structurally distinct amphiphilic copolymers of 2-hydroxyethyl methacrylate (HEMA) and styrene have been examined with angle-dependent X-ray photoelectron spectroscopy (XPS) and static secondary ion mass spectrometry (SIMS). The phase-separated block copolymer made by anionic living polymerization, HSH-A50, showed significant surface enrichment of styrene. The outermost 2-3 A appeared to be approximately 100% styrene, with the styrene concentration decreasing to its bulk value at a depth of approximately 50 A from the surface. However, HEMA was detected in the outer 20 A of this copolymer. The presence of HEMA in the surface region implies this copolymer may undergo significant restructuring when hydrated in a hydrophilic environment (as opposed to the hydrophobic environment in which the sample was prepared and analyzed). The phase-separated block copolymer made by telechelic coupling of free radical polymerized functionalized oligomers, HSH-B60, showed only slight styrene enrichment at the surface. Both HEMA and styrene were detected at all sampling depths, including the outermost surface layer, consistent with the presence of discrete HEMA and styrene domains at the copolymer surface. Since both components are already present at the surface under hydrophobic conditions, the degree of restructuring this copolymer may undergo upon hydration should be minor. The random HEMA--styrene copolymer made by conventional free radical initiation techniques, HS-RAN50, had a surface composition that was similar to the bulk composition and independent of depth, as expected for a homogeneously mixed copolymer film.

Biocompatible Materials↗
PubMed1991

Small unilamellar liposomes from mixed natural and polymeric phospholipids: stability and susceptibility to phospholipase A2.

The concept of the uncorkable liposome composed of phase-separated mixtures of a polymerized phospholipid and an enzymically digestible phospholipid has been investigated, using small unilamellar vesicles composed of mixtures of (polymerized) dienoylphosphatidylcholine (DENPC) and dimyristoylphosphatidylcholine (DMPC). Mixed liposomes, even those containing only 10% DENPC, were much more stable than DMPC liposomes, as indicated by the release of entrapped [3H]inulin or [14C]glucose. DMPC liposomes released entrapped solute on exposure to phospholipase A2, whereas mixed vesicles were resistant. The results are compared with those of an earlier study on monolayers of similar compositions. It is concluded that the liposomes, like the monolayers, are phase-mixed, and that uncorkable liposomes cannot be constructed from the phospholipid mixture employed. It is proposed that, until further experimental evidence is produced, the enzymatically uncorkable liposome must be regarded as a theoretical construct.

Carbon Radioisotopes↗
PubMed1990

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy.

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Mixed monolayers of natural and polymeric phospholipids: structural characterization by physical and enzymatic methods.

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

Chemical Phenomena↗
PubMed1990

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. II: Surface characterization and in vitro assessments.

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), as well as heparin-coated glass beads and tubes were evaluated for the amounts and activities of surface-immobilized heparin. Because the amphiphilic copolymer system is thermodynamically predicted to demonstrate low-energy phase enrichment on the surfaces of air-cast films, studies were also undertaken to understand the in vitro results. Solvent-cast copolymer films have a heterogeneous microphase-separated structure according to transmission electron micrographs. Wilhelmy plate contact angle analysis indicates significant surface restructuring occurs upon hydration. Attenuated total reflectance infrared spectroscopy studies of the desiccated and hydrated films at two different sampling depths show compositional heterogeneity as a function of depth, as well as near surface restructuring allowing surface enrichment of the high-energy segments following contact with water. Significant concentrations of heparin are detected on the surface of these coatings by toluidine blue assays. In addition, a portion of the surface-bound heparin maintains its original bioactivity as determined by recalcification times, thrombin times, and Factor Xa assays. These substrates were also tested for platelet adhesion and activation reactions in vitro using polymer-coated beads in rabbit platelet-rich plasma. Heparinized polymers promoted low levels of platelet adhesion and serotonin release. Surface concentrations of heparin from bioactivity assays were then correlated with platelet adhesion and the extent of platelet release to assess the efficacy of this heparin-immobilized copolymer as a blood-compatible material or coating.

Animals↗
PubMed1990

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. III: Surface and bulk compositional differences.

Previously observed bioactivity of poly(dimethylsiloxane)-poly(ethylene oxide)-heparin (PDMS-PEO-Hep) triblock copolymers has prompted studies of the surface and bulk character of this copolymer using angular-dependent electron spectroscopy for chemical analysis (ADESCA), static secondary mass spectroscopy (SIMS), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Because the low-energy PDMS phase dominates surfaces of this copolymer when solvent cast under air or vacuum conditions, attempts were made to explain surface restructuring and rearrangements induced in hydrated or aqueous environments that permit surface accessibility and bioactivity of heparin moieties. Based on comparisons with PDMS, PEO, and heparin homopolymers, PEO/heparin blends, and an unheparinized PDMS-PEO diblock copolymer, PDMS-PEO-heparin demonstrates both phase-mixed and phase-separated regions in DSC analysis. During annealing cycles above the Tg values of the copolymer constituents, phase-mixed regions become increasingly phase separated and PEO enriched. TGA analysis confirmed the presence block copolymer constituents and presented evidence of intermolecular segmental interactions, hence phase-mixing in the copolymers. ADESCA analysis indicates that the outer 5 A of both the PDMS-PEO and PDMS-PEO-Hep copolymers is essentially pure PDMS. However, significant amounts of PEO are detected 5 to 20 A below the surface. Static SIMS also detects the presence of PDMS at the surfaces of the PDMS-PEO and PDMS-PEO-Hep copolymers. Compositional models based on ADESCA, SIMS, and DSC data are presented for desiccated and hydrated copolymer surfaces.

Biocompatible Materials↗
PubMed1989

In vitro and ex vivo platelet interactions with hydrophilic-hydrophobic poly(ethylene oxide)-polystyrene multiblock copolymers.

Hydrophilic-hydrophobic multiblock copolymers synthesized from telechelic oligomers of poly(ethylene oxide) (PEO) and polystyrene (PS) have been used to study the influence of hydrophilic and hydrophobic balance on interfacial interactions of these surfaces with blood components. In vitro coagulation assays show no inherent ability of these amphiphilic surfaces to affect contact activation or coagulation factors. In vitro platelet adhesion and release reactions from rabbit platelet-rich plasma were shown to be greatest on Biomer and PS homopolymer surfaces and least on cross-linked PEO surfaces, with the PEO-PS block copolymers demonstrating intermediate responses. These same substrates were tested in a new low-flow, low-shear arterio-artery shunt system in rabbits. Whole blood occlusion times were not a direct function of hydrophilic content as both PEO and PS homopolymers and Biomer showed short occlusion times, while PEO-PS block copolymers prolonged occlusion times considerably, depending on composition. Overall, results suggest that PEO-PS block copolymers promote unique whole blood responses in contrast to homopolymer and Biomer controls which are more complex than direct correlations to bulk hydrophilic and hydrophobic contents.

Anastomosis, Surgical↗
PubMed1988

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. I. Synthesis and characterization.

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), and heparin (PDMS-PEO-Hep) have been prepared via a series of coupling reactions using functionalized prepolymers, diisocyanates, and derivatized heparins. All intermediate steps of the synthesis yield quantifiable products with reactive end-groups, while the final products demonstrate bioactive, covalently bound heparin moieties. Due to the solvent systems required, commercial sodium heparin was converted to its benzyltrimethyl ammonium salt to enhance its solubility. The same procedure was applied to heparin degraded by nitrous acid in order to covalently couple it in solutions with the semitelechelic copolymers. As might be expected, this derivatization reduces the apparent bioactivity of the heparin. However, preliminary findings suggest that the bioactivity can be restored by reforming the heparin sodium salt.

Biocompatible Materials↗
PubMedDate not supplied

Membrane microstructural templates for enzyme domain formation.

Soluble proteins can spontaneously self-organize into two-dimensional domains at membrane interfaces, given sufficient mobility and specificity to membrane-localized ligands. The authors' recent results studying interfacial domain formation of the membrane-active enzyme, phospholipase A2, indicate that lateral phase separation of heterogeneous membrane mixtures creates anionic templates of specific morphology onto which the enzyme deposits, forming large protein assemblies. Selective removal of membrane components (lysolipid or fatty acid) produces different enzyme interfacial responses and domain morphologies. This leads to the conclusion that complex chemical and physical interactions laterally in the lipid membrane interface as well as between bound protein molecules play a role in organizing protein structures.

Binding Sites↗
PubMedDate not supplied

In vivo nonthrombogenicity of heparin immobilized polymer surfaces.

The authors developed two different methods to immobilize heparin on polymer surfaces. One method involves in situ heparin immobilization on a segmented polyurethane urea (Biomer) surface via hydrophilic poly(ethylene oxide) (PEO, Mn = 4,000) spacers. The other method uses PEO/poly(dimethylsiloxane) (PDMS) block co-polymer and heparin covalently linked in a block co-polymer system (PEO-PDMS-Hep). These surfaces have demonstrated high heparin bioactivity in vitro and excellent blood compatibility in in vitro-ex vivo experiments. This report evaluates the long-term in vivo blood compatibility of these heparin immobilized surfaces. Vascular grafts (6 mm ID, 7 cm in length) were fabricated with Biomer, and heparin was immobilized in situ with PEO spacers (B-PEO4K) and coated on their luminal surfaces with PEO-PDMS-Hep. Biomer and PEO (Mn = 4,000) grafted Biomer (B-PEO4K) were used as controls. The grafts were implanted in the abdominal aorta of dogs and retrieved at 3 months or when graft occlusion was suspected. Retrieved grafts were evaluated with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM measured the thickness of the adsorbed protein layer on the surface and the protein distribution (albumin, fibrinogen, and IgG) visualized by an immunogold method. All heparin immobilized grafts were patent at 3 months, whereas Biomer and B-PEO4K grafts occluded within 1 month. SEM pictures of heparin immobilized surfaces after 3 months demonstrated minimal platelet adhesion and activation without detectable fibrin formation. Heparin immobilized surfaces showed a thin protein layer (300-600 A) even after 3 months, with high concentrations of albumin and IgG and less fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗