Date of Award

6-1-1994

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Stephen R. Grant

Second Advisor

Rafael Alvarez

Third Advisor

Paula Sumstom

Abstract

Xiaoqiang, Kang., Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway. Doctor of Philosophy (Biomedical Sciences), June, 1994, 150 pp., 4 tables, 36 illustrations, bibliography, 212 titles. The cDNA for human interleukin-8 (IL8) was subcloned from a bacterial source into the eukaryotic baculoviral vector expression system. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from SF9 cells following infection of a recombinant virus harboring the full-length IL-8 structural gene. Recombinant human interleukin-8 was purified (>600 fold) to homogeneity using preparative HPLC. The rhIL-8 preparation retained all of the physical, immunological, and biochemical properties of the natural product (monocyte-derived IL-8). Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production. Biochemical mechanisms that mediate IL-8 receptor-stimulated activities are poorly understood. In this study, I have explored the intracellular mechanism(s) induced by IL-8 in differentiated HL-60 cells. IL-8 induced a rapid and transient activation of phospholipase A2 in differentiated HL-60 cells. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. The IL-8 stimulated-arachidonic acid release was pertussis toxin and phospholipase A2 inhibitor sensitive, and protein kinase C independent. In contrast to another neutrophil chemotactic factor, fMLP, IL-8 did not stimulate the activation of phospholipase C and phospholipase D. When comparing the phosphorylation events induced by IL-8 and fMLP, I found that these two chemotactic factors triggered different protein phosphorylation profiles. Tyrosine phosphorylation of proteins was not detected following IL-8 stimulation in HL-60 cells. However, IL-8 stimulated the rapid autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaM kinase II). These results strongly suggest that the IL-8 receptor is closely coupled to the activation of PLA2 and that CaM kinase II is an integral component of IL-8 receptor signal pathway.

Comments

Xiaoqiang, Kang., Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway. Doctor of Philosophy (Biomedical Sciences), June, 1994, 150 pp., 4 tables, 36 illustrations, bibliography, 212 titles. W 4 K16I 1994

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