Biomedical subjects

Patrick S Stayton

Publications and source records attributed to Patrick S Stayton.

At least 19 recordsLinked to original sources

PubMed2006

Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals.

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.

Computer Simulation↗

PubMed2006

Thermodynamics of statherin adsorption onto hydroxyapatite.

Statherin is a salivary protein that inhibits the nucleation and growth of hydroxyapatite crystals in the supersaturated environment of the oral cavity. The thermodynamics of adsorption of statherin onto hydroxyapatite crystals have been characterized here by isothermal titration calorimetry and equilibrium adsorption isotherm analysis. At 25 degrees C, statherin adsorption is characterized by an exothermic enthalpy of approximately 3 kcal/mol that diminishes to zero at approximately 25% surface coverage. The initial heat of statherin adsorption increases with temperature, displaying a positive heat capacity change of 194 +/- 7 cal K(-)(1) mol(-)(1) at 25 degrees C. The heat of adsorption during this initial phase is strongly dependent on the buffer species, and from the differential heats of buffer ionization, it can be calculated that approximately one proton is taken up by the crystal or protein upon adsorption. The free energy of adsorption is dominated at all coverages by a large positive entropy (>or=23 cal K(-)(1) mol(-)(1)), which may be partially due to the loss of organized water that hydrates the protein and the mineral surface prior to adsorption. These results are interpreted using a two-site model for adsorption of statherin onto the hydroxyapatite crystals.

Adsorption↗

PubMed2006

A solid-state NMR study of the dynamics and interactions of phenylalanine rings in a statherin fragment bound to hydroxyapatite crystals.

Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, (13)C rotating frame relaxation measurements, as well as direct detection of correlations between (13)C spins on protein side chains and (31)P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide's N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.

Amino Acid Sequence↗

PubMed2006

Cooperative hydrogen bond interactions in the streptavidin-biotin system.

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.

Biotin↗

PubMed2006

Poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers that respond sharply to temperature and pH.

Temperature- and pH-sensitive random copolymers of N-isopropylacrylamide (NIPAAm) and propylacrylic acid (PAA) were prepared using the reversible addition fragmentation chain transfer (RAFT) polymerization method. The lower critical solution temperatures (LCSTs) (or phase separation temperatures) of the NIPAAm-co-PAA copolymer solutions were measured by the cloud-point method. At slightly acidic conditions, the LCST decreased with increase in PAA content, which suggests that the hydrophobic propyl group of PAA has a greater influence on the LCST than the polar carboxylic acid group at those conditions. An increase of pH led to a significant increase in LCST of the copolymers due to the ionization of the -COOH group. The LCSTs were studied as a function of copolymer composition over the pH range from 5.0 to 7.0. Because the pK(a) of the polymers can be tuned to fall close to neutral pH, these polymer compositions can be designed to have phase transitions triggered near physiological pH or at slightly acidic pH values that fall within acidic gradients found in biology. The NIPAAm-co-PAA copolymers thus display tunable properties that could make them useful in a variety of molecular switching and drug delivery applications where responses to small pH changes are relevant.

Acrylamides↗

PubMed2006

PEG-cross-linked heparin is an affinity hydrogel for sustained release of vascular endothelial growth factor.

An affinity-based controlled release system for growth factors having heparin-binding domains was prepared using a cross-linked heparin gel. The heparin gel was made by reacting hydrazide-functionalized heparin (Hep-ADH) with the N-hydroxysuccinimidyl ester of poly(ethylene glycol)-bis-butanoic acid (SBA-PEG-SBA). The degree of cross-linking could be controlled by defining the stoichiometry of hydrazide modification and the PEG cross-linker addition. The release of vascular endothelial growth factor (VEGF) was characterized as a heparin-binding growth factor. VEGF was directly injected into the heparin gel and the loaded VEGF displayed a slow, controlled release over 3 weeks with little initial burst phase. The biological activity of the released VEGF was measured with a proliferation assay utilizing human umbilical vein endothelial cells. The released VEGF maintained its biological activity at all time points investigated. The heparin gel with loaded VEGF was implanted sub-cutaneously in the dorsal region of mice. A significantly increased density of the endothelial cell marker platelet endothelial adhesion molecule (PECAM-1) was observed in histological specimens of the tissues surrounding the implanted gel.

Animals↗

PubMed2005

Dual-affinity avidin molecules.

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.

Animals↗

PubMed2005

A REDOR NMR study of a phosphorylated statherin fragment bound to hydroxyapatite crystals.

Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (15N) spin in the lysine side chain and two phosphorus-31 (31P) spins, at least one of which must be from the surface phosphates of the HAP.

Crystallization↗

PubMed2005

Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. "Smart" pH-responsive, membrane-destabilizing polymers present a new approach to shuttling therapeutic molecules past the endosomal membrane and into the cytoplasm of targeted cells. This report describes the use of a functionalized monomer, pyridyl disulfide acrylate (PDSA), to develop pH-responsive, membrane-destabilizing, and glutathione-reactive polymers by copolymerization with several pH-responsive and hydrophobic monomers. The activity of the carriers is described as a function of (a) increasing the length of the hydrophobic alkyl group substituted onto the pH-responsive monomer and (b) the incorporation of a hydrophobic monomer such as butyl acrylate (BA) on the pH sensitivity and membrane-destabilizing activity of new polymer compositions. The membrane-destabilizing activity of different polymer compositions was evaluated as a function of pH and polymer concentration using the red blood cell (RBC) hemolysis assay. Hemolysis results show that the increase in the hydrophobic character of the polymer backbone results in a shift in the pH sensitivities and an increase in the membrane-destabilizing activity. Results show that the observed hemolytic activities and pH sensitivity profiles could be designed across a range that matches the properties needed for enhancing the cytoplasmic delivery of macromolecular therapeutic.

Acrylates↗

PubMed2005

A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells.

The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.

Acrylates↗

PubMed2005

Design and construction of highly stable, protease-resistant chimeric avidins.

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Amino Acid Sequence↗

PubMed2005

Rational design of composition and activity correlations for pH-sensitive and glutathione-reactive polymer therapeutics.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. "Smart" pH-sensitive, membrane-destabilizing polymers present an attractive approach to shuttle therapeutic molecules past the endosomal membrane and into the cytoplasm of targeted cells. This report describes the use of a new functionalized monomer, pyridyl disulfide acrylate (PDSA), to develop pH-sensitive, membrane-destabilizing, and glutathione-reactive polymers by copolymerization with several pH-sensitive and hydrophobic monomers. The activity of the carriers is described as a function of (a) the influence of increasing the length of the hydrophobic alkyl group substituted onto the pH-sensitive monomer and (b) of the effect of incorporating a hydrophobic monomer such as butyl acrylate (BA) on the pH sensitivity and membrane-destabilizing activity of new polymer compositions. The membrane-destabilizing activity of different polymer compositions was evaluated as a function of pH and polymer concentration using the red blood cells (RBC) hemolysis assay. Hemolysis results show that the increase in the hydrophobic character of polymer backbone results in a shift in the pH sensitivity profile and an increase in the membrane-destabilizing activity. Results show that the observed hemolytic activities and pH sensitivity profiles could be designed across a range that matches the properties needed for drug carriers to enhance the cytoplasmic delivery of therapeutic cargos.

Acrylates↗

PubMed2005

Design and development of polymers for gene delivery.

The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.

Animals↗

PubMed2005

Poly(propylacrylic acid)-mediated serum stabilization of cationic lipoplexes.

The serum instability associated with cationic lipoplexes represents one of the major obstacles for the in vivo delivery of nonviral gene therapy vectors. Recently, we have shown that poly(propylacrylic acid) (PPAA), a pH-sensitive polyanionic polymer, can significantly improve the in vitro serum stability of DOTAP lipoplexes and enhance transfection (Cheung et al., Bioconjug. Chem. 12, 906 (2001)). We investigated this serum-stabilizing effect provided by PPAA using methods to identify the specific serum proteins that interact with DOTAP/DNA and DOTAP/DNA/PPAA lipoplexes and determined their modes of interaction with these lipoplexes. Studies showed that only low-density lipoprotein (LDL) caused significant decondensation of DNA from lipoplexes lacking PPAA, but that fully condensed DNA was retained within lipoplexes incorporating PPAA. Another major factor in the loss of transfection activity was due to the reduced cellular uptake of DOTAP lipoplexes upon exposure to serum, with bovine serum albumin (BSA) and high-density lipoprotein (HDL) acting as major contributors to this reduction in vector internalization. In contrast, lipoplexes containing PPAA maintained high levels of uptake into cells in the presence of these proteins. Transfection results generally concurred with the mechanistic studies, suggesting that maintaining effective cellular delivery of intact lipoplexes in the presence of serum proteins is important to retain high transfection efficiencies. These results indicate that the addition of PPAA as a ternary component in DOTAP lipoplexes can overcome some of the serum-related deficiencies encountered with these lipoplexes to provide efficient transfection.

Acrylates↗

PubMed2005

Semi-interpenetrating network of poly(ethylene glycol) and poly(D,L-lactide) for the controlled delivery of protein drugs.

We have prepared a semi-interpenetrating network (IPN) of poly(ethylene glycol) dimethacrylate (PEGDMA) with entrapped poly(D,L-lactide) (PLA) using photochemical techniques. These IPNs were developed for the controlled delivery of protein drugs such as growth factors. The PEG component draws water into the network, forming a hydrogel within the PLA matrix, controlling and facilitating release of the protein drug, while the PLA component both strengthens the PEG hydrogel and enhances the degradation and elimination of the network after the protein drug is released. The rate and extent of swelling and the resultant protein release kinetics could be controlled by varying the PEG/PLA ratio and total PLA content. These IPNs were prepared using a biocompatible benzyl benzoate/benzyl alcohol solvent system that yields a uniform, fine dispersion of the protein throughout the PEG/PLA IPN matrix. IPNs composed of high molecular mass PLA and lower PEG/PLA ratios exhibited lower equilibrium swelling ratios. The release of bovine serum albumin (BSA), a model protein, from these IPNs was characterized by a large initial burst, regardless of the PEG/PLA ratio, due to the entrapment of residual solvent within the network. Microparticles of the PEG/PLA IPNs were also prepared using a modified Prolease strategy. Residual solvent removal was significantly enhanced using this process. The microparticles also exhibited a significant reduction in the initial burst release of protein. Mixtures of different compositions of PEG/PLA microparticles should be useful for the delivery of a variety of protein drugs with different release kinetics from any tissue-engineering matrix.

Acrylates↗

PubMed2005

Monocyte activation on polyelectrolyte multilayers.

The adherence and activation of primary human monocytes was investigated on a polyelectrolyte multilayer film containing hyaluronic acid (HA) and poly-L-lysine (PLL). The sequential layer-by-layer deposition of the multilayer film was characterized by surface plasmon resonance. Eight alternating bilayers displayed an effective thickness of 16.15 nm with a total polymer coverage of 2.10 microg/cm2. For cell studies, HA-PLL multilayers were constructed on tissue culture polystyrene (TCPS) substrates and characterized by time of flight second ion mass spectrometry (ToF-SIMS) analysis. Principal component analysis of the ToF-SIMS spectra resolved no significant difference in surface chemistry between PLL-terminated and HA-terminated multilayer surfaces. Monocyte adhesion on PLL- and HA-terminated surfaces was measured by the lactate dehydrogenase assay and showed a significant decrease in cell adhesion after 24 h incubation. Cell viability measured by Live/Dead fluorescent staining showed significant cell death in the adherent cell population over these 24 h. Tumor necrosis factor-alpha (TNF-alpha) production, a measure of monocyte activation, was quantified by ELISA and normalized to the number of adherent monocytes. The activation of monocytes on PLL-terminated and HA-terminated surfaces was nearly identical, and both surfaces had TNF-alpha levels that were 8-fold higher than TCPS. These results demonstrate that sufficient PLL had diffused into the surface layer to direct monocyte adherence and to induce cytokine activation and cell death on the HA-terminated multilayer films. The diffusion of the second multilayer component to the coating surface should, thus, be taken into account in the design of polyelectrolyte-based biomaterial coating strategies.

Cell Adhesion↗

PubMed2005

Smart polymeric carriers for enhanced intracellular delivery of therapeutic macromolecules.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. Researchers have examined several approaches, including fusogenic proteins and protein transduction domains, to enhance the intracellular delivery of the therapeutic cargo. This review summarises efforts to develop 'smart' pH-sensitive and membrane-destabilising polymers that can shuttle therapeutic peptide, protein and nucleic acid molecules past the endosomal membrane into the cytoplasm of targeted cells. Several classes of 'smart' non-degradable polymeric carriers have been developed that have proved effective both in vitro and in vivo in enhancing the cytoplasmic delivery of a variety of therapeutic molecules.

Animals↗

PubMed2004

Hyaluronic acid grafting mitigates calcification of glutaraldehyde-fixed bovine pericardium.

Pathologic calcification is the leading cause of the clinical failure of glutaraldehyde-fixed bovine pericardium used in bioprosthetic valves. A novel surface modification of glutaraldehyde fixed bovine pericardium was carried out with high molecular weight hyaluronic acid (HA). HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional groups onto the HA backbone. Glutaraldehyde-fixed bovine pericardium (GFBP) was modified by grafting this HA to the free aldehyde groups on the tissue via the hydrazide groups. Following a 2-week subcutaneous implantation in osteopontin (OPN)-null mice, the calcification of HA-modified bovine pericardium was drastically reduced (by 84.5%) compared to positive controls (tissue without HA-modification) (p = 0.005). The calcification-mitigating effect of HA surface modification was also confirmed by microscopic analysis of explanted tissue stained with Alizarin Red S for calcium.

Animals↗