C.G. dos Remedios

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.

Fluorescence Depolarization of Actin Filaments in Reconstructed Myofibers: The Effect of S1 or pPDM-S1 on Movements of Distinct Areas of Actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle

AIMS We examined cardiomyocyte apoptosis in chronic heart failure (HF) and its possible link to elevated wall stress. METHODS AND RESULTS Moderate HF was produced in sheep by sequential coronary microembolization. Six months later, the animals remained in a stable compensated haemodynamic state of HF. Apoptosis of cardiomyocytes in left ventricles was verified using Western blotting based on increased expression of: the apoptosis-associated death receptor Fas (1.5-fold); its ligand (FasL, 2.0-fold); and an upstream protease caspase-8 (2.7-fold) as well as its active cleavage peptide, p20 (5.6-fold). Previously we have reported the elevated expression of caspase-3 in the same animal model. The occurrence of apoptotic cardiomyocytes (0.3%) was quantified by TUNEL assays. Haemodynamic analysis indicated that ventricular dilatation, without wall thickening, caused a 2-fold increase in LV wall stress which, together with LV end-diastolic pressure, was linearly correlated with expression of Fas/FasL. Immunohistochemical studies localized FasL and caspase-8 to intercalated discs, suggesting that wall stress may play a role in initiating cardiomyocyte apoptosis. CONCLUSION Apoptosis of cardiomyocytes in chronic HF is associated with increased wall stress, which may be responsible for the activation of a Fas/FasL and caspase-8 interaction in the region of intercalated discs.

Genomics, proteomics and bioinformatics of human heart failure.

Unraveling the molecular complexities of human heart failure, particularly end-stage failure, can be achieved by combining multiple investigative approaches. There are several parts to the problem. Each patient is the product of a complex set of genetic variations, different degrees of influence of diets and lifestyles, and usually heart transplantation patients are treated with multiple drugs. The genomic status of the myocardium of any one transplant patient can be analysed using gene arrays (cDNA- or oligonucleotide-based) each with its own strengths and weaknesses. The proteins expressed by these failing hearts (myocardial proteomics) were first investigated over a decade ago using two-dimensional polyacrylamide gel electrophoresis (2DGE) which promised to resolve several thousand proteins in a single sample of failing heart. However, while 2DGE is very successful for the abundant and moderately expressed proteins, it struggles to identify proteins expressed at low levels. Highly focused first dimension separations combined with recent advances in mass spectrometry now provide new hope for solving this difficulty. Protein arrays are a more recent form of proteomics that hold great promise but, like the above methods, they have their own drawbacks. Our approach to solving the problems inherent in the genomics and proteomics of heart failure is to provide experts in each analytical method with a sample from the same human failing heart. This requires a sufficiently large number of samples from a sufficiently large pool of heart transplant patients as well as a large pool of non-diseased, non-failing human hearts. We have collected more than 200 hearts from patients undergoing heart transplantations and a further 50 non-failing hearts. By combining our expertise we expect to reduce and possibly eliminate the inherent difficulties of each analytical approach. Finally, we recognise the need for bioinformatics to make sense of the large quantities of data that will flow from our laboratories. Thus, we plan to provide meaningful molecular descriptions of a number of different conditions that result in terminal heart failure.

Effects of Gd(III) on G-actin: Inhibition of polymerization of G-actin and activation of myosin ATPase activity by Gd-G-actin

Summary When a critical amount of Gd(III) is added to G-actin before attempting to polymerize this protein, there is a total inhibition of polymer formation as judged by changes in the reduced viscosity. This inhibition can be reversed by removal of Gd(III). Such Gd-treated G-actin is able to activate myosin ATPase activity in the same way as untreated G-actin. This loss of polymerization together with the preservation of the myosin binding site may prove valuable in attempts to obtain crystals of G-actin.

Responses of skeletal muscle fibres to lanthanide ions. Dependence of the twitch response on ionic radii.

A study of the effects of the lanthanide series of ions on toad skeletal muscle fibres reveals that they a) inhibit the twitch response, b) exhibit a marked dependence on ionic radius, and c) apparently exert their effects on the sarcolemma.

Binding stoichiometry of gadolinium to actin: its effect on the actin-bound divalent cation.

Abstract Using 45 Ca 2+ and 153 Gd 3+ we studied the effects of binding the lanthanide ion, gadolinium, to skeletal muscle G-actin. Gd 3+ can specifically displace 6–7 Ca 2+ from their binding sites on actin. Furthermore, a total of 6–7 Gd 3+ can be shown to bind to actin, and this result is not affected by the subsequent addition of polymerizing quantities of KCl. We conclude that Gd 3+ binds only to the Ca 2+ -binding sites of actin. The number of these Gd 3+ sites closely corresponds to the known number of high and low affinity sites for divalent cations such as Ca 2+ .

Linker-free covalent thermophilic β-glucosidase functionalized polymeric surfaces

The surface immobilization of enzymes is important for many biomedical and industrial applications; however, the choice of enzyme and immobilization method for best performance is often difficult. This paper demonstrates that Plasma Immersion Ion Implantation (PIII) provides high enzyme coverage and rapid covalent attachment (within the first minute) of incubation, and shows how this can be used to control the composition of the attached layer from an enzyme mixture. The use of a thermophilic enzyme combined with covalent immobilization viaPIII-treatment significantly improves β-glucosidase activity. β-glucosidase is an important rate-limiting enzyme in the production of cellulosic biofuel and is useful in many other industrial applications. β-glucosidase immobilized on the PIII-treated polystyrene surface demonstrated significant improvements in reaction rate and glucose yields compared to the enzyme on the untreated surface. A thermophilic β-glucosidase immobilized with our PIII-treatment method shows an average glucose production rate more than twenty-fold higher, over five uses, compared to the commercially-available β-glucosidase immobilized by physisorption after purification.

Cluster of differentiation antibody microarrays on plasma immersion ion implanted polycarbonate

Plasma immersion ion implantation (PIII) modifies the surface properties of polymers, enabling them to covalently immobilize proteins without using linker chemistry. We describe the use of PIII treated polycarbonate (PC) slides as a novel platform for producing microarrays of cluster of differentiation (CD) antibodies. We compare their performance to identical antibody microarrays printed on nitrocellulose-coated glass slides that are currently the industry standard. Populations of leukocytes are applied to the CD microarrays and unbound cells are removed revealing patterns of differentially immobilized cells that are detected in a simple label-free approach by scanning the slides with visible light. Intra-slide and inter-slide reproducibility, densities of bound cells, and limits of detection were determined. Compared to the nitrocellulose-coated glass slides, PIII treated PC slides have a lower background noise, better sensitivity, and comparable or better reproducibility. They require three-fold lower antibody concentrations to yield equivalent signal strength, resulting in significant reductions in production cost. The improved transparency of PIII treated PC in the near-UV and visible wavelengths combined with superior immobilization of biomolecules makes them an attractive platform for a wide range of microarray applications.

An actin-myosin functional assay for analysis of smooth muscle (anti-microfilament) autoantibodies in human plasma.

The detection of serum autoantibodies to smooth muscle (SMA) on rodent gastric mucosa by indirect immunofluorescence (IIF) has long been an immunodiagnostic marker for autoimmune hepatitis type 1 (AIH-1). The reactive antigenic moieties are cytoskeletal proteins which include polymeric F-actin as judged by the staining of microfilaments of tissue by IIF. However, their specificity for actin in AIH-1 can be and usually is uncertain. Using an in vitro functional assay, we compared the effects of Fab fragments of immunoglobulin (IgG) prepared from SMA-positive plasma from two patients with the effects of Fabs from 10 healthy subjects. Fabs are incorporated into an assay where actin (the putative antigen) activates skeletal muscle heavy meromyosin (HMM) ATPase activity. The data from these functional assays provide new insights into the significance of anti-microfilament assays in the diagnosis, and perhaps also pathogenesis, of AIH-1.