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Biomedical subjects

P E Dawson

Publications and source records attributed to P E Dawson.

At least 19 recordsLinked to original sources
PubMed1998

Chemical synthesis of human protein S thrombin-sensitive module and first epidermal growth factor module.

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR: residues 47-75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76-116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluron ium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys85-Cys102) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys80-Cys93 and Cys104-Cys113) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys85-Cys102). CD studies revealed 54% of beta-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% beta-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway.

Amino Acid Sequence↗
PubMed1997

Total chemical synthesis of enzymatically active human type II secretory phospholipase A2.

Human group II secretory phospholipase A2 (sPLA2) is an enzyme found in the alpha granules of platelets and at inflammatory sites. Although its physiological function is unclear, sPLA2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA2 activities, we developed a total chemical synthesis of sPLA2 by chemical ligation of large unprotected peptides. The synthetic segments PLA2-(1-58)-alphaCOSCH2COOH and PLA2-(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly58 and Cys59. The 124-residue polypeptide product (mass: 13,920 +/- 2 Da) was folded to yield one major product (mass: 13,905 +/- 1 Da), the loss of 15 +/- 3 Da reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA2 showed alpha-helix, beta-structure, and random coil contents consistent with those found in the crystal structure of sPLA2. Synthetic sPLA2 had kcat and Km values identical to those of recombinant sPLA2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA2, like recombinant sPLA2, inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca2+, and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca2+. Thus, synthetic sPLA2 is a phospholipid-independent anticoagulant like recombinant or natural sPLA2. This study demonstrates that chemical synthesis of sPLA2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA2 analogs for structure-activity studies of anticoagulant, lipolytic, or inflammatory activities.

Amino Acid Sequence↗
PubMed1996

A classification system for occlusions that relates maximal intercuspation to the position and condition of the temporomandibular joints.

Interarch occlusal relationships are defined by temporomandibular joint (TMJ) position. Determination of the most physiologic joint position is a logical prerequisite for occlusal analysis. Existing classification systems for occlusion do not consider TMJ position or condition when relating the mandibular arch to the maxillary arch or the range of adaptive changes that can affect the position of the condyles or influence long-term occlusal stability. If the relationship between occlusion and TMJ position is as important as many clinicians believe, condylar position must be defined precisely as an essential control in any clinical study that purports to evaluate the relationship between occlusion and any masticatory system disorder to include temporomandibular disorders. This article presents a new classification system that defines the relationship between maximal intercuspation and the position and condition of the TMJs. The classification uses guidelines that are specific enough to be consistent and verifiably reproducible. A recently introduced term, "adapted centric posture," is used in this classification to distinguish deformed TMJs that have remodeled or adapted to a conformation that can comfortably accept maximal loading. This classification is necessary because deformed but adapted joints may within certain conditions function with the same degree of comfort as intact, properly aligned condyle disk assemblies in centric relation.

Adaptation, Physiological↗
PubMed1996

Probing the chemical basis of binding activity in an SH3 domain by protein signature analysis.

BACKGROUND: Modifying the covalent structure of a protein is an effective empirical route to probing three-dimensional structure and biological function. Here we describe a combinatorial protein chemistry strategy for studying structure-activity relationships in proteins. Our approach (termed 'protein signature analysis') involves functional selection from an array of self-encoded protein analogs prepared by total synthesis, coupled to a simple chemical readout that unambiguously identifies the modified proteins in the resulting active and inactive populations. RESULTS: Protein signature analysis was used to study the interaction of the amino-terminal SH3 domain from the cellular adaptor protein c-Crk with its cognate proline-rich peptide, C3G. Using a functional selection assay, the qualitative effects of scanning a series of synthetic analog units through the amino-acid sequence of the SH3 domain were evaluated. The analog units were designed to alter both amino-acid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp 150 in the SH3 domain is essential for ligand binding and that changes in the structure of the polypeptide backbone can also result in loss of binding activity. CONCLUSIONS: These chemical studies have provided new insight into how ligand binding is related to the covalent structure of the SH3 domain. Protein signature analysis is a powerful and conceptually novel way of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's chemical structure with the practical convenience of combinatorial synthesis.

Amino Acid Sequence↗
PubMed1996

Template-directed ligation of peptides to oligonucleotides.

BACKGROUND: Oligonucleotide-peptide conjugates have several applications, including their potential use as therapeutic agents. We developed a strategy for the chemical ligation of unprotected peptides to oligonucleotides in aqueous solution. The two compounds are joined via a stable amide bond in a template-directed reaction. RESULTS: Peptides, ending in a carboxy-terminal thioester, were converted to thioester-linked oligonucleotide-peptide intermediates. The oligonucleotide portion of the intermediate binds to a complementary oligonucleotide template, placing the peptide in close proximity to an adjacent template-bound oligonucleotide that terminates in a 3' amine. The ensuing reaction results in the efficient formation of an amide-linked oligonucleotide-peptide conjugate. CONCLUSIONS: An oligonucleotide template can be used to direct the ligation of peptides to oligonucleotides via a highly stable amide linkage. The ligation reaction is sequence-specific, allowing the simultaneous ligation of multiple oligonucleotide-peptide pairs.

Amides↗
PubMed1996

A new attachment system for removable partial dentures.

How a removable partial denture (RPD) attaches to its abutment teeth is the most important aspect of partial denture design. The reason is obvious: Many critical requirements for optimal design are dependent on the way the removable segment is related and secured to the abutment teeth. To appreciate the importance of the attachment design, it is necessary to understand several principles of overall RPD design. State-of-the-art RPD design is often the most effective means for achieving long-term maintainable health of the remaining teeth. Even weakened teeth with compromised bone support can, with good design, often be used effectively as abutments for RPDs while benefitting from the removable partial.

Denture Design↗
PubMed1995

New definition for relating occlusion to varying conditions of the temporomandibular joint.

Centric relation is the accepted term for defining the condylar axis position of intact, completely seated, properly aligned condyle-disk assemblies. However, some structurally deformed temporomandibular joints may function comfortably, even though they do not fulfill the requirements for centric relation. A wide range of temporomandibular disorders from partial to complete disk derangements with or without reduction may adapt to a conformation that permits the joints to comfortably accept maximal compressive loading by the elevator muscles. There has been no accepted terminology to define the condition or position of such joints. The purpose of this article is to define a new term, adapted centric posture, and to explain its rationale and how it is determined. Verification of successful adaptation is an important step in diagnosis, because it rules out structural intracapsular disorders as a source of orofacial pain and establishes responsible guidelines for initiation of occlusal treatment or prosthetic dentistry. It also establishes a much needed terminology for more specific description of temporomandibular joint position and condition for clinical research on the relationship between occlusion and the temporomandibular joints.

Adaptation, Physiological↗
PubMed1994

Synthesis of proteins by native chemical ligation.

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Amino Acid Sequence↗
PubMed1994

Effects of hypothermia upon endothelial cells: mechanisms and clinical importance.

The endothelial cell is vital in the regulation of blood vessel wall structure, vasomotor tone, and thrombogenicity. Hypothermic temperatures alter both the physiological and biochemical dynamics of endothelial cells. However, there has been no systematic investigation of the influence of cold temperatures upon endothelial cell biology. This review summarizes the current clinical areas of interests, identifies the problems, and addresses the fundamental requirement for further research in endothelial cell cryobiology.

Animals↗
PubMed1993

Cytotoxicity of amphotericin B for fibroblasts in human heart valve leaflets.

The cytotoxicity of amphotericin B (Fungizone, containing deoxycholate) was investigated for human heart valve leaflet fibroblasts. Leaflets were obtained from human aortic and pulmonic valves and incubated in culture medium containing amphotericin B. Upon completion of incubation, some leaflet sets were analyzed immediately, and others were cryopreserved and stored below -135 degrees C. Quantitative fibroblast viability assays were performed. The results can be summarized by consideration of the data obtained from autoradiographic analysis of [3H]proline incorporation into collagen. Incubation with 10 micrograms/ml amphotericin B at 37 degrees C resulted in approximately 11% loss of fibroblast viability. After cryopreservation, the leaflets incubated with amphotericin B experienced an additional 42% loss of fibroblast viability. These results indicate that use of amphotericin B, in this form, is ill advised for treatment of human heart valves prior to cryopreservation.

Amphotericin B↗
PubMed1992

Effects of storage temperature on viable bioprosthetic heart valves.

Long-term in vivo success of bioprosthetic allografts is dependent upon retention of cellular functions, such as protein synthesis. The purpose of the experiments presented in this report was to determine the storage conditions necessary for retention of protein synthetic functions in human allograft heart valve leaflets. Tissue viability was assessed by measurement of tritiated-glycine incorporation into proteins. Comparison of short-term (less than 3 month)- and long-term (1 and 2 years)-cryopreserved heart valve leaflet storage in a liquid nitrogen freezer below -135 degrees C demonstrated preservation of fibroblast protein synthesis. In contrast, storage in a mechanical freezer at -80 degrees C resulted in a time-dependent loss of fibroblast protein synthesis. There was no statistically significant effect on protein synthesis in leaflets stored for 1 week at 4 degrees C compared to control cryopreserved liquid nitrogen-stored leaflets. After 2 weeks of 4 degrees C storage leaflet protein synthesis declined significantly to 15% that of cryopreserved controls. These results demonstrate that liquid nitrogen storage of valve bioprostheses is required for long-term preservation of cellular functions.

Bioprosthesis↗