PDGF and cardiovascular disease

Platelet-derived growth factor (PDGF) was identified in a search for serum factors that stimulate smooth muscle cell (SMC) proliferation. During the development of lesions of atherosclerosis that can ultimately lead to vessel occlusion, SMC are stimulated by inflammatory factors to migrate from their normal location in the media. They accumulate within the forming lesion where they contribute to lesion expansion by proliferation and deposition of extracellular matrix. Different genetic manipulations in vascular cells combined with various inhibitory strategies have provided strong evidence for PDGF playing a prominent role in the migration of SMC into the neointima following acute injury and in atherosclerosis. Other activities of PDGF identified in vivo suggest additional functions for PDGF in the pathogenesis of cardiovascular disease.     

The CXC motif: a functional mimic of protein disulfide isomerase

Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding. Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI. This peptide has a thiol acid dissociation constant (pK(a) = 8.7) that is lower than that of glutathione. These attributes endow the CGC peptide with substantial disulfide isomerization activity. Escherichia coli thioredoxin (Trx) utilizes the active site sequence Cys-Gly-Pro-Cys (CGPC; E degrees ' = -270 mV) to effect disulfide reduction. Removal of the proline residue from the Trx active site yields a CGC active site with a greatly destabilized disulfide bond (E degrees ' >or= -200 mV). The DeltaP34 variant retains high conformational stability and remains a substrate for thioredoxin reductase. In contrast to the reduced form of the wild-type enzyme, the reduced form of DeltaP34 Trx has disulfide isomerization activity, which is 25-fold greater than that of the CGC peptide. Thus, the rational deletion of an active site residue can bestow a new and desirable function upon an enzyme. Moreover, a CXC motif, in both a peptide and a protein, provides functional mimicry of PDI.     

Individual and geographical variation in display behaviour of male harbour seals in Scotland

Studying variations in behaviour at the individual or population level enables insight into the reproductive strategies within a species. We examined individual and geographical variation in the vocal and dive behaviour of male harbour seals, Phoca vitulina, which is associated with aquatic mating. This display behaviour was recorded in the Moray Firth, Scotland, from July 1994 to 1997, and in Orkney, Scotland, during July 1998. One vocalization type was apparent in the Moray Firth and two in Orkney. Time parameters (total and pulse duration) varied between males in the population in the Moray Firth. We used both frequency and time parameters in a discriminant analysis, which showed that 73.2% of individual male vocalizations could be correctly classified; 94.6% of male vocalizations from the Moray Firth and Orkney could be correctly classified according to their geographical areas. Therefore, vocal variation was greater between geographical areas than between individuals. No individual variation was apparent between dive and surface interval durations. However, individuals varied significantly in the percentage of short surface intervals. Male harbour seals showed substantial variability in the parameters affecting their vocal and dive behaviour during the mating season. We suggest that these variations may be indicative of adaptations to varying environmental challenges influencing the reproductive strategies of discrete populations. Copyright 2000 The Association for the Study of Animal Behaviour.     

Genetic selection for dissociative inhibitors of designated protein-protein interactions

Many biological processes rely on protein-protein interactions. These processes include signal transduction, cell cycle regulation, gene regulation, and viral assembly and replication. Moreover, many proteins and enzymes manifest their function as oligomers. We describe here an efficient means to sift through large combinatorial libraries and identify molecules that block the interaction of target proteins in vivo. The power of this approach is demonstrated by the identification of nine-residue peptides from a combinatorial library that inhibit the intracellular dimerization of HIV-1 protease. Fewer than 1 in 106 peptides do so. In vitro biochemical analyses of one such peptide demonstrate that it acts by dissociating HIV-1 protease into monomers, which are inactive catalysts. Inhibition is enhanced further by dimerizing the peptide. This approach enables the facile identification of new molecules that control cellular processes.     

Genetic selection reveals the role of a buried, conserved polar residue

The burial of nonpolar surface area is known to enhance markedly the conformational stability of proteins. The contribution from the burial of polar surface area is less clear. Here, we report on the tolerance to substitution of Ser75 of bovine pancreatic ribonuclease (RNase A), a residue that has the unusual attributes of being buried, conserved, and polar. To identify variants that retain biological function, we used a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic activity. Cell growth at 30 degrees C, 37 degrees C, and 44 degrees C correlated with residue size, indicating that the primary attribute of Ser75 is its small size. The side-chain hydroxyl group of Ser75 forms a hydrogen bond with a main-chain nitrogen. The conformational stability of the S75A variant, which lacks this hydrogen bond, was diminished by DeltaDeltaG = 2.5 kcal/mol. Threonine, which can reinstate this hydrogen bond, provided a catalytically active RNase A variant at higher temperatures than did some smaller residues (including aspartate), indicating that a secondary attribute of Ser75 is the ability of its uncharged side chain to accept a hydrogen bond. These results provide insight on the imperatives for the conservation of a buried polar residue.     

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fcγ receptors (FcγR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil FcγRs were examined with a specific FcγR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of FcγRIIa and FcγRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative FcγR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.