Mark E. Andracki

Lipoprotein Lipase Binds to Low Density Lipoprotein Receptors and Induces Receptor-mediated Catabolism of Very Low Density Lipoproteins in Vitro

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of plasma triglycerides, promotes binding and catabolism of triglyceride-rich lipoproteins by various cultured cells. Recent studies demonstrate that LPL binds to three members of the low density lipoprotein (LDL) receptor family, including the LDL receptor-related protein (LRP), GP330/LRP-2, and very low density lipoprotein (VLDL) receptors and induces receptor-mediated lipoprotein catabolism. We show here that LDL receptors also bind LPL and mediate LPL-dependent catabolism of large VLDL with Sf 100-400. Up-regulation of LDL receptors by lovastatin treatment of normal human foreskin fibroblasts (FSF cells) resulted in an increase in LPL-induced VLDL binding and catabolism to a level that was 10-15-fold greater than in LDL receptor-negative fibroblasts, despite similar LRP activity in both cell lines. This indicates that the contribution of LRP to LPL-dependent degradation of VLDL is small when LDL receptors are maximally up-regulated. Furthermore studies in LRP-deficient murine embryonic fibroblasts showed that the level of LPL-dependent degradation of VLDL was similar to that in normal murine embryonic fibroblasts. LPL also promoted the internalization of protein-free triglyceride emulsions; lovastatin-treatment resulted in 2-fold higher uptake in FSF cells, indicating that LPL itself could bind to LDL receptors. However, the lower induction of emulsion catabolism as compared with native VLDL suggests that LPL-induced catabolism via LDL receptors is only partially dependent on receptor binding by LPL and instead is primarily due to activation of apolipoproteins such as apoE. A fusion protein between glutathione S-transferase and the catalytically inactive carboxyl-terminal domain of LPL (GST-LPLC) also induced binding and catabolism of VLDL. However GST-LPLC was not as active as native LPL, indicating that lipolysis is required for a maximal LPL effect. Mutations of critical tryptophan residues in GST-LPLC that abolished binding to VLDL converted the protein to an inhibitor of lipoprotein binding to LDL receptors. In solid-phase assays using immobilized receptors, LDL receptors bound to LPL in a dose-dependent manner. Both LPL and GST-LPLC promoted binding of VLDL to LDL receptor-coated wells. These results indicate that LPL binds to LDL receptors and suggest that the carboxyl-terminal domain of LPL contributes to this interaction.

Rotary Culture Enhances Pre-osteoblast Aggregation and Mineralization

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

Preparation of intramolecularly cross-linked hemoglobins

Publisher Summary This chapter discusses the preparation of intramolecularly cross-linked hemoglobins. Hemoglobin (Hb), although normally a tetramer, readily undergoes dissociation to form αβ dimers. By cross-linking the molecule intramolecularly, this process can be blocked. This has become important both for basic structure-function studies of Hb and for the development of Hb derivatives useful as blood substitutes. Preparation of mixed metal and valency hybrids has made it possible to isolate stable species representing intermediate ligation states of Hb; these have yielded important new insights into the linkage between ligand binding and the structural and energetic changes that underlie cooperativity. Cross-linking is also important from the standpoint of developing Hb derivatives to serve as blood substitutes. The αβ dimers having a molecular weight of only 32,000 are readily filtered from the circulation by the kidneys. As a result, unmodified Hb has a very short plasma half-life, and the massive hemoglobinuria that results poses the risk of renal injury. It is essential to cross-link the molecule intramolecularly to prevent this from occurring.

Prolongation of the Intravascular Retention of Hemoglobin Modified with a Long-Chain Fatty Acid Derivative

To develop hemoglobin (Hb) derivatives with an increased circulatory half-life, Hb was chemically modified with long chain fatty acid analogs. One compound, sodium 1-hexadecyl 6-(2-iodoacetamido)hexyl phosphate, specifically modified the Cys-93 beta residues of human hemoglobin (HbA) as determined by sulfhydryl titration analysis. The resulting modified Hb derivative, FAHbA, was isolated and was shown to have a two-fold longer circulatory half-life than native HbA in a rat low-dose acute transfusion model.