Plasma treatment of polyurethane coating for improving endothelial cell growth and adhesion.

The advantage of helium plasma treatment in enhancing endothelial cell growth and adhesion on polyurethane film coated on glass substrate is demonstrated with experimental data. Human coronary artery endothelial cell (HCAE) growth and attachment was studied on (1) bare glass substrate, used as control, (2) coated glass, with and without helium plasma treatment and (3) collagen-treated polyurethane-coated glass substrates. The untreated polyurethane film surface was rough (RMS = 690 nm) and highly hydrophobic (contact angle theta = 90 degrees). Cell growth on the untreated polyurethane surface was poor (cell concentration approximately 3750/cm2) compared to glass surface (cell concentration approximately 17 665/cm2). The atmospheric helium plasma treatment of the polyurethane film resulted in oxidation of the surface, a slight increase in roughness (RMS = 735 nm) and a significant drop in hydrophobicity (contact angle theta = 79 degrees). The critical surface tension (gamma c) of polyurethane film was also increased by 2 dynes/cm due to helium plasma treatment. These changes resulted in enhanced HCAE cell growth in polyurethane film (cell concentration approximately 16 230/cm2) compared to the untreated polyurethane film. The cell growth was also comparable to cell growth on a glass surface (17 665/cm2) and the collagen-treated polyurethane film surfaces (cell concentration approximately 21 645/cm2), respectively. Moreover, the strength of cell attachment on a plasma-treated surface (cell retention R = 89%) under laminar flow was significantly higher than that on a glass surface (R = 71%). While the collagen-treated polyurethane surface had the highest number of HCAE cells, the cell adhesion was found to be poor (R = 42%) compared to that of a plasma-treated surface. Thus, the overall performance of the plasma-treated polyurethane film surface on endothelial cell growth was better than other substrates studied here.

Corrosion resistance of polyurethane-coated nitinol cardiovascular stents.

Corrosion of metal stents implanted inside an artery can have two adverse effects: (1) tissue reaction and possible toxic effects from the metal ions leaching out of the stent, and (2) loss of mechanical strength of the stent caused by corrosion. The corrosion resistance of Nitinol (Nickel-Titanium) stents and its modulation with different film thickness of polymer coating was studied against an artificial physiological solution using a Potentiostat/Galvanostat and an electrochemical corrosion cell. The corrosion rate decreased rapidly from 275 microm/year for an uncoated surface down to less than 13 microm/year for a 30 microm thick polyurethane coating. Stainless steel (316L) and Nitinol both contain potentially toxic elements, and both are subject to stress corrosion. Minimization of corrosion can significantly reduce both tissue reaction and structural degradation.

Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis.

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus.

The generation of an autoimmune response against islet beta-cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta-cells themselves. Reactive oxygen species (ROS) have been implicated in the beta-cell destruction which leads to type 1 diabetes and may modify beta-cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta-cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro-insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell-free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.

Soluble urokinase receptor promotes cell adhesion and requires tyrosine-92 for activation of p56/59(hck).

The urokinase plasminogen activator receptor (uPAR) plays an important role in the migration of leukocytes. It occurs as a membrane-bound form that contains a glycosylphosphatidylinositol (GPI) anchor and also as a soluble form (suPAR) that lacks the GPI anchor. Recently, a sequence of amino acids, SRSRYLE, within the receptor has been found to become unmasked on uPA binding or chymotrypsin cleavage. Exposure of the epitope results in the activation of p56/p59(hck) kinase and chemotaxis of myelomonocytic cells. Using an epitope-tagged suPAR molecule, we found that both three-domain and two-domain suPAR promote the adhesion of differentiated THP-1 cells to fibronectin and vitronectin, indicating that suPAR can modify cell adhesion as well as cell migration. In addition, we found that the amino acid sequence RYLE, within the chemotactic peptide, is conserved across species and that alanine substitution of Tyr 92 decreased the ability of the peptide to activate p56/59(hck).

Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase.

BACKGROUND: Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed beta and beta', are conserved throughout nature. The beta subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the beta subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the beta subunit of RNA polymerase. To this end, the beta structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector. RESULTS: Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the beta polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo. CONCLUSIONS: These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of beta may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF-Tu in stringent control.

Physical mapping of a collection of Mael-generating amber mutations in the beta gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation.

We report the fine mapping of 55 of our 95 amber mutations in the beta gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e. CTAG, where the amber codon is underlined). [The full data are reported in Supplementary Publication SUP 50181 (12 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8-10.] The CTAG mutations, which have been positioned to within approx. 9-60 bp, are distributed almost along the entire length of the rpoB gene, the one exception being the interval 400-499. The lack of amber fragments for mutations within the 5' approx. 265 codons suggests lability of the extreme N-terminal region; further potential destabilizing 'signals' may be present in the non-conserved 'spacer' regions. The locations of four of the eleven rpoB amber mutations that are strongly polar on expression of the downstream rpoC gene have been determined through a combination of MaeI mapping, PCR amplification and DNA sequencing. Surprisingly, one such mutant carries two tandem CTAG sites but is viable with three of the nonsense suppressors tested. These polar amber sites define three different amino acids (Gln-31, Gln-83 and Trp-183) that fall within three sequence-conservation blocks in the N-terminal region. Six of the MaeI/Am (where MaeI/Am is an amber mutation generating a MaeI restriction site) rpoB alleles (Gln-83, Gln-276, Gln-327, Gln-618, Gln-649 and Trp-183) have been used to generate small in-frame deletions (31-100 codons) within conserved and non-conserved regions of the beta gene, and the properties of these deletion variants were assessed in vivo. The smallest deletion reported in this study removes 31 amino acids from the middle of a region common to the eubacterial/chloroplast subgroup of beta homologues, and our results strongly suggest beta(deltaQ618-Q649) is assembled into a holoenzyme form capable of transcriptional initiation in vivo.