Gordon A. Vehar

Gene Expression Profiling in an in Vitro Model of Angiogenesis

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Production of Recombinant Coagulation Proteins

Genetic engineering emerged from the field of molecular biology and has developed into a technology that has impacted a wide variety of biological disciplines. This ability to identify, manipulate, and characterize genes has resulted in dramatic increases in our understanding of gene and protein structure, the molecular basis of hereditary disease, posttranslational processing, and evolution of an ever expanding list of proteins. This information has revolutionized biochemical research by revealing the molecular structure of both genes and proteins for the system under study. In addition to the information/knowledge benefit of genetic engineering, the promise of therapeutic applications of proteins produced by recombinant DNA technology has recently become a reality. Regulatory approval has been achieved for recombinant therapeutic preparations of a hepatitis vaccine, human insulin, human growth hormone, and for alpha interferon. Several additional recombinant proteins are at various stages within the regulatory approval process and should receive approval within the next few years. The availability of plentiful supplies of recombinant forms of human proteins is revolutionizing the pharmaceutical industry and in the process is having a beneficial effect upon public health. Hemostasis is one of the fields wherein genetic engineering has recently made a major impact. Prior to 1980, the trace amounts of many of the proteins of both the coagulation cascade and the fibrinolytic system had prevented detailed characterization. The availability of cDNA clones has allowed the determination of the complete structure of even the most trace of the plasma proteins and enabled the identification and characterization of the human genes for these proteins. The resulting body of information has defined at the molecular level the various proteins involved in hemostatis. The hereditary bleeding disorders of the hemostatic system are of major clinical significance. Three diseases account for the vast majority of afflicted individuals: deficiency of factor VIII (hemophilia A or classic hemophilia); deficiency of factor IX (hemophilia B); and deficiency of von Willebrand factor (von Willebrand’s disease). The ability to probe gene structure is, for the first time, allowing an understanding of the genetic basis of these diseases at the molecular level. In addition, the ability to construct plasmids directing the expression of human proteins in cell lines capable of large scale culture could have a major impact upon future treatment regimens. The clinical success of recombinant tissue plasminogen activator in the treatment of myocardial infarction is a promising example of the tremendous power of the technology to produce pharmaceutical products. In this review, the potential of biotechnology to

The Human Factor Viii Protein

Publisher Summary This chapter focuses on the human factor VIII protein. Factor VIII is apparently secreted as a single chain polypeptide of 2332 amino acids containing 25 potential asparagine-linked glycosylation sites. As isolated from plasma, the protein has undergone varying degrees of proteolytic degradation depending on the species and/or starting material. The homology of a composite plastocyanin protein sequence with the 100 amino acid carboxy-terminal portion of each of the three domains of factor VIII is shown diagrammatically in the chapter. The plastocyanins are proteins of plant origin that function in electron transfer reactions associated with photosynthesis. Plastocyanins contain copper centers in “blue” or type 1 configurations. Such copper centers are characterized by an absorption band near 600 nm with an unusually high extinction coefficient, an electron paramagnetic resonance spectrum with a small hyperfine splitting constant, and a relatively high redox potential when compared to that of the Cu(II)/Cu(I) couple in solution.