Neil J. Nosworthy

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces

Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices.

A Comparison of Covalent Immobilization and Physical Adsorption of a Cellulase Enzyme Mixture

This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented.

Attachment of horseradish peroxidase to polytetrafluorethylene (teflon) after plasma immersion ion implantation

The aim of this work was to investigate the potential of polytetrafluorethylene (PTFE) as a surface for biologically active protein attachment. A plasma immersion ion implantation (PIII) treatment was applied to PTFE to produce an activated surface for the functional attachment of the enzyme, horseradish peroxidase (HRP). Fourier transform infrared-attenuated total reflectance spectra show oxidation and carbonization of the surface layer as a function of ion fluence. The PIII treatment increases by threefold the amount of attached HRP and the activity of HRP on the modified surface is about seven times higher than that on an untreated PTFE surface. This result indicates that the PIII surface modification improves both the polymers protein binding capacity and its ability to retain the protein in a bioactive state.

Cofilin and DNase I Affect the Conformation of the Small Domain of Actin

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 A (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 A), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 A) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells.

Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics.

The multiple causes and multiple consequences of mammalian heart failure make it an attractive proposition for analysis using gene array technology, especially where the failure is idiopathic in nature. However, gene arrays also hold potential artefacts, particularly when gene expression levels are low, and where changes in expression levels are modest. Also, at present, the number of genes available on arrays is not large enough to prevent potential sampling deficiencies. Thus, it may not be wise to place too much reliance on quantitative interpretations of gene array data. Also, recently doubts were raised about the qualitative reliability of array genes. Electrophoretic methods are slow, cumbersome and complex but they can provide confirmation that the trends and numbers arising from the new gene arrays are reliable. In this overview, we compare gene array data with data from protein activity assays such as zymograms, Western blots, two‐dimensional electrophoresis, and immunohistochemistry. Similar or complementary data from the same heart tissues analyzed by either microarrays or macroarrays can be reassuring to those interested in reliable molecular analyses of normal and failing hearts. Similar principles will apply to other tissues and cells.

A New Surface for Immobilizing and Maintaining the Function of Enzymes in a Freeze-Dried State

We describe a new surface produced by plasma treatment for immobilizing proteins in the dry state. The need for surfaces suitable for immobilizing proteins is increasing because of demand for microarray diagnostic services, biosensors, and chemical processing. Storage of surface attached proteins in the dry state offers benefits of long shelf life, protection from proteases, easier transportation and convenient storage. In this work, we produced plasma-modified polyethylene surfaces and tested them using two important enzymes for which convenient functional assays are available, namely, horseradish peroxidase and catalase. Over 80% of the function of horseradish peroxidase is retained after freeze-drying, and this function is unaltered after 4 months of storage at 4 degrees C on the treated polyethylene surface. The factors important for maintenance of surface attached enzyme stability were (1) plasma immersion ion implantation (PIII) treatment of the surface, (2) freeze-drying with sucrose in the buffer solution, (3) dry storage with desiccant, and (4) maintaining the freeze-dried protein at a reduced temperature. Other than sucrose, no other additives are needed.

Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts

Vnd/NK-2 protein was detected in 11 neuroblasts per hemisegment in Drosophila embryos, 9 medial and 2 intermediate neuroblasts. Fragments of DNA from the 5′-flanking region of the vnd/NK-2 gene were inserted upstream of an enhancerless βgalactosidase gene in a P-element and used to generate transgenic fly lines. Antibodies directed against Vnd/NK-2 and β-galactosidase proteins then were used in double-label experiments to correlate the expression of β-galactosidase and Vnd/NK-2 proteins in identified neuroblasts. DNA region A, which corresponds to the −4.0 to −2.8-kb fragment of DNA from the 5′-flanking region of the vnd/NK-2 gene was shown to contain one or more strong enhancers required for expression of the vnd/NK-2 gene in ten neuroblasts. DNA region B (−5.3 to −4.0 kb) contains moderately strong enhancers for vnd/NK-2 gene expression in four neuroblasts. Hypothesized DNA region C, whose location was not identified, contains one or more enhancers that activate vnd/NK-2 gene expression only in one neuroblast. These results show that nucleotide sequences in at least three regions of DNA regulate the expression of the vnd/NK-2 gene, that the vnd/NK-2 gene can be activated in different ways in different neuroblasts, and that the pattern of vnd/NK-2 gene expression in neuroblasts of the ventral nerve cord is the sum of partial patterns.

Effect of low molecular weight additives on immobilization strength, activity, and conformation of protein immobilized on PVC and UHMWPE.

Horseradish peroxidase (HRP) was immobilized onto both plasticized and unplasticized polyvinylchloride (PVC) and ultrahigh molecular weight polyethylene (UHMWPE). Plasma immersion ion implantation (PIII) in a nitrogen plasma with 20 kV bias was used to facilitate covalent immobilization and to improve the wettability of the surfaces. The surfaces and immobilized protein were studied using attenuated total reflection infrared (ATR-IR) spectroscopy and water contact angle measurements. Protein elution on exposure to repeated sodium dodecyl sulfate (SDS) washing was used to assess the strength of HRP immobilization. The presence of low molecular weight components (plasticizer, additives in solvent, unreacted monomers, adsorbed molecules on surface) was found to have a major influence on the strength of immobilization and the conformation of the protein on the samples not exposed to the PIII treatment. A phenomenological model considering interactions between the low molecular weight components, the protein molecule, and the surface is developed to explain these observations.

The role of ATP, ADP and divalent cations in the formation of binary and ternary complexes of actin, cofilin and DNase I

Actin is the major cytoskeletal protein of virtually all eukaryotic cells. Actin assembly/disassembly is involved in a variety of cellular processes and actin‐binding proteins are essential in regulation of the pool of actin monomers. Cofilin and DNase I are actin‐binding proteins, which form both binary (actin‐DNase 1, cofilin‐actin) and ternary (cofilin‐actin‐DNase I) complexes with actin. Here we use native gel electrophoresis to examine the roles of ATP, ADP, Ca2+ and Mg2+ in the formation of these complexes as well as on the ability of actin to self‐assemble. Conditions which favour actin polymerisation are: ATP (no Me2+) ≥ ADP (no Me2+) > ADP‐Ca2+ = ADP‐Mg2+ > ATP‐Mg2+ > ATP‐Ca2+. Preferential conditions for the formation of the binary actin‐cofilin complex are: ADP‐Mg2+ ≥ ADP‐Ca2+ ≫ ATP‐Ca2+ ≈ ATP‐Mg2+ ≈ ADP‐No Me2+ ≈ ATP‐No Me2+. Actin forms a very tight complex with DNase I in the order: ATP‐Ca2+ ≥ ATP‐Mg2+ ≈ ADP‐Mg2+ ≈ ADP‐Ca2+ ≥ ADP‐(no Me2+) > ATP‐(no Me2+). Effectively, the complex does not form in the presence of ATP and the absence of free Me2+. Finally, the conditions which favour the formation of a ternary complex of cofilin‐actin‐DNase I resemble the actin‐DNase I, namely: ATP‐Ca2+ ≈ ADP‐Ca2+ ≈ ADP‐Mg2+ ≈ ATP‐Mg2+ ≈ ADP (no Me2+) > ATP‐(no Me2+).