No abstract

Summary Background The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. Objectives Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. Methods ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without amutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. Results In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. Conclusions A region of the VWF promoter between −2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.

No abstract

Biomineralized uniform and well-organized calcium carbonate microspheres were synthesized for enzyme immobilization, and the immobilized enzyme was successfully stabilized. The physicochemical parameters of calcium carbonate were studied using scanning electron microscopy with energy-dispersive X-ray spectroscopy, particle size analysis, X-ray diffraction analysis, Fourier-transform infrared spectroscopy, and surface area measurement. Additionally, Barrett-Joyner-Halenda adsorption/desorption analysis showed that the calcium carbonate microspheres provided efficient mesopore space for enzyme loading. As a model enzyme, carboxyl esterase (CE) was entrapped and then cross-linked to form an enzyme structure. In this aggregate, the cross-linked enzymes cannot leach out from mesopores, resulting in enzyme stability. The hydrolytic activities of the free and cross-linked enzymes were analyzed over broad temperature and pH ranges. The cross-linked enzyme displayed better activity than the free enzyme. Furthermore, the immobilized CE was found to be stable for more than 30 days, preserving 60% of its initial activity even after being reused more than 10 times. This report is expected to be the first demonstration of a stabilized cross-linked enzyme system in calcium carbonate microspheres, which can be applied in enzyme catalyzed reactions involved in bioprocessing, bioremediation, and bioconversion.