Biomedical subjects

John A M Ramshaw

Publications and source records attributed to John A M Ramshaw.

13 recordsLinked to original sources

PubMed2006

MR three-dimensional molecular imaging of intramural biomarkers with targeted nanoparticles.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Animals↗

PubMed2005

Effect of deamidation on stability for the collagen to gelatin transition.

Deamidation of amide residues, Asn and Gln, in collagen occurs during the manufacture of B-type gelatin and could affect the performance of B-type gelatins as it may affect the refolding of triple-helical junctional domains that are formed during gelation. Host-guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide, where the X- and Y-positions of the guest peptide are varied, have been used to examine the effect of changing Asn to Asp and Gln to Glu on triple-helix stability. This paper reports the stability of host-guest peptides containing the guest triplets Gly-Ala-Asn, Gly-Asn-Ala, Gly-Asn-Lys, Gly-Gln-Ala, Gly-Glu-Glu, and Gly-Leu-Glu. In combination with previous data, these now provide 15 pairs of peptides in which the effect of deamidation can be compared. These comparisons show that the deamidation of Asn to Asp, regardless of whether it occurred in either the X- or Y-position, always gave a stabilizing effect; deamidation of Gln in the X-position also led to an increase in stability. In contrast, deamidation of Gln in the Y-position was quite distinct, leading to destabilization. The higher observed frequency of Gln in the Y-position compared with other amides may account for the slight destabilization of collagen following deamidation.

Amides↗

PubMed2005

Prediction of collagen stability from amino acid sequence.

An algorithm was derived to relate the amino acid sequence of a collagen triple helix to its thermal stability. This calculation is based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm. However, predicted T(m) values are significantly higher than experimental values when there are strings of oppositely charged residues or concentrations of like charges near the terminus. Application of the algorithm to collagen sequences highlights regions of unusually high or low stability, and these regions often correlate with biologically significant features. The prediction of stability from sequence indicates an understanding of the major forces maintaining this protein motif. The use of highly favorable KGE and KGD sequences is seen to complement the stabilizing effects of imino acids in modulating stability and may become dominant in the collagenous domains of bacterial proteins that lack hydroxyproline. The effect of single amino acid mutations in the X and Y positions can be evaluated with this algorithm. An interactive collagen stability calculator based on this algorithm is available online.

Algorithms↗

PubMed2005

Electrostatic interactions involving lysine make major contributions to collagen triple-helix stability.

Important stabilizing features for the collagen triple helix include the presence of Gly as every third residue, a high content of imino acids, and interchain hydrogen bonds. Host-guest peptides have been used previously to characterize triple-helix propensities of individual residues and Gly-X-Y triplets. Here, comparison of the thermal stabilities of host-guest peptides of the form (Gly-Pro-Hyp)3-Gly-X-Y-Gly-X'-Y'-(Gly-Pro-Hyp)3 extends the study to adjacent tripeptide sequences, to encompass the major classes of potential direct intramolecular interactions. Favorable hydrophobic interactions were observed, as well as stabilizing intrachain interactions between residues of opposite charge in the i and i + 3 positions. However, the greatest gain in triple-helix stability was achieved in the presence of Gly-Pro-Lys-Gly-Asp/Glu-Hyp sequences, leading to a T(m) value equal to that seen for a Gly-Pro-Hyp-Gly-Pro-Hyp sequence. This stabilization is seen for Lys but not for Arg and can be assigned to interchain ion pairs, as shown by molecular modeling. Computational analysis shows that Lys-Gly-Asp/Glu sequences are present at a frequency much greater than expected in collagen, suggesting this interaction is biologically important. These results add significantly to the understanding of which surface ion pairs can contribute to protein stability.

Amino Acid Motifs↗

PubMed2005

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.

Animals↗

PubMed2005

Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle-urothelial cell interactions.

The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then harvested for evaluation of matrix contraction. Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAM may contain some key inductive factors that serve to promote important bladder cell-cell and cell-matrix interactions.

Animals↗

PubMed2004

Stepwise construction of triple-helical heparin binding sites using peptide models.

The specific localization of the asymmetric form of acetylcholinesterase (AChE) in neuromuscular junctions results from the interaction of its collagen-like tail with heparan sulfate proteoglycans in the synaptic basal lamina. This interaction involves two heparin binding consensus sequences of the form XBBXB, where B is a basic residue, located in the triple-helical collagen tail: GRKGR for the N-terminal site and GKRGK for the C-terminal site. To explore the basis of the higher heparin affinity seen for the C-terminal site vs. the N-terminal site, two homologous series of (Gly-Xaa-Yaa)(8) peptides were constructed to model these triple-helical binding sites. Individual tripeptide units from each heparin binding site were introduced in a stepwise fashion into a Gly-Pro-Hyp framework, until the consensus sequence and its surrounding triplets were recreated. As each additional triplet from the binding site is inserted to replace a host Gly-Pro-Hyp triplet, the triple-helix stability decreases, and the drop in thermal stability is close to that expected if each Gly-X-Y triplet contributed independently to global stability. CD spectroscopy and calorimetry show the stability of these AChE model peptides is increased by addition of heparin, confirming binding to heparin, and the lack of significant enthalpy change indicates the binding is largely electrostatic in nature. Displacement assays measure the strength of the peptide-heparin interaction, and indicate an inverse correlation between the peptide ability to bind heparin and its thermal stability. The model peptides for the C-terminal binding site show a greater heparin affinity than the peptide models for the N-terminal binding site only when residues surrounding the consensus sequence are included.

Amino Acid Sequence↗

PubMed2004

Collagen-hydroxyapatite composite prepared by biomimetic process.

A novel bone graft substitute comprising a porous, collagenous scaffold was biomimetically coated with hydroxyapatite using a simulated body fluid solution chemistry method. The scaffold had a porosity of approximately 85%, with pore sizes between 30 microm and 100 microm. Glutaraldehyde vapor was used to stabilize the collagenous scaffold, giving a significantly increased thermal stability over an unstabilized scaffold, as shown by differential scanning calorimetry. A thin layer (<10 microm) of crystalline hydroxyapatite was deposited onto the stabilized collagenous scaffold by soaking the collagenous construct in simulated body fluid in the presence of calcium silicate glass. The presence of crystalline hydroxyapatite was confirmed by X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. In vitro cytotoxicity testing of the composite construct using L-929 fibroblasts (ISO 10993-5) and rabbit periosteal cells revealed a cytocompatible material that supported cellular attachment and proliferation.

Animals↗

PubMed2003

Triple-helix propensity of hydroxyproline and fluoroproline: comparison of host-guest and repeating tripeptide collagen models.

Peptide models have proved important in defining the structural features of the collagen triple-helix. Some models are based on multiple repeats of a given tripeptide unit, while a host-guest design includes an individual tripeptide unit substituted within a constant repeating Pro-Hyp-Gly framework. In the present study, proline, hydroxyproline, and fluoroproline residues are incorporated in X- or Y-positions of a guest triplet in the host-guest peptide design. All host-guest peptides, including Hyp-Pro-Gly, formed stable triple-helices, even though a triple-helix cannot be formed by (Hyp-Pro-Gly)10. The order of stability Pro-Hyp-Gly > Pro-Pro-Gly > Hyp-Pro-Gly remains the same in all models, while the Pro-Flp-Gly is very stabilizing in a repeating context but destabilizing in a host-guest context. The range of thermal stabilities and calorimetric enthalpies is very small among the five host-guest peptides, consistent with the concept that the effect of one Xaa-Yaa-Gly tripeptide unit in the host-guest system would be less than the much larger variations when there are 10 repeating units. However, a simple additive model based on host-guest peptides predicts a greater stability than experimentally observed. The difference in stability contributions of the same tripeptide unit in host-guest versus repeating tripeptide systems illustrates the impact of sequence environment on stability, and factors that play a role include ring puckering as a consequence of electron inductive effects, residual monomer structure, and native state hydration networks.

Amino Acid Sequence↗

PubMed2003

Fluorescence determination of tryptophan side-chain accessibility and dynamics in triple-helical collagen-like peptides.

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.

Amino Acid Sequence↗

PubMed2002

Peptide investigations of pairwise interactions in the collagen triple-helix.

Pairwise interactions have been studied for the major secondary structures in proteins. The present work extends the characterization of interactions between side-chains to the context of a collagen triple-helix. In this study, the most frequent Gly-X-Y tripeptide sequences in collagen are characterized in terms of interchain interactions between non-imino acid X and Y residues, through the use of host-guest peptides and statistical frequency analysis. Stabilities predicted on the basis of additivity show good agreement with experimental values for almost half of the peptides, indicating a lack of interaction. A small number of peptides have a stability lower than predicted, while a larger number are more stable than expected. Of all triplets containing residues of opposite charge, only Gly-Lys-Asp and Gly-Arg-Asp exhibit stabilizing electrostatic interactions, and these pairs are found together preferentially in collagens. Repulsion of like charges is observed in Gly-Arg-Lys, Gly-Lys-Arg, and Gly-Glu-Asp sequences, and a small degree of hydrophobic stabilization was observed for the Gly-Leu-Leu guest triplet. The data reported here help clarify basic principles of triple-helix stability. In addition, the experimentally determined stabilities of the tripeptide units found most frequently in collagens constitute a database useful for predicting triple-helix stability in peptides, collagens and other triple-helix-containing proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Collagen fibril formation in a wound healing model.

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.

Animals↗

PubMedDate not supplied

Evaluation for collagen products for cosmetic application.

Collagen is an important component for cosmetic formulation, where it is an effective natural humectant with high substantivity. Commercial collagen preparations have a wide range of properties. In the present study, various techniques have been used to examine three distinct commercial collagens that illustrate the range of properties that are available. The usefulness of the various techniques for assessing collagen quality and batch-to-batch variation is discussed. The results indicate that there are several simple, cheap, and effective methods, such as gel electrophoresis, that provide excellent information on collagen quality. The appropriate selection of tests allows informed decisions on the choice of which collagen preparation to use to provide the desired functionality and shelf life of a formulation.

Amino Acids↗