In Vivo Serial MR Imaging of Magnetically Labeled Endothelial Progenitor Cells Homing to the Endothelium Injured Artery in Mice

Background: Emerging evidence of histopathological analyses suggests that endothelial progenitor cells (EPCs) play an important role in vascular diseases. Neointimal hyperplasia can be reduced by intravenous transfusion of EPCs after vascular injury in mice. Therefore, it would be advantageous to develop an in vivo technique that can explore the temporal and spatial migration of EPCs homing to the damaged endothelium noninvasively. Methodology/Principal Findings: The left carotid common artery (LCCA) was injured by removal of endothelium with a flexible wire in Kunming mice. EPCs were collected by in vitro culture of spleen-derived mouse mononuclear cells (MNCs). EPCs labeling was carried out in vitro using Fe2O3-poly-L-lysine (Fe2O3-PLL). In vivo serial MR imaging was performed to follow-up the injured artery at different time points after intravenous transfusion of EPCs. Vessel wall areas of injured artery were computed on T2WI. Larger MR signal voids of vessel wall on T2WI was revealed in all 6 mice of the labeled EPC transfusion group 15 days after LCCA injury, and it was found only in 1 mouse in the unlabeled EPC transfusion group (p = 0.015). Quantitative analyses of vessel wall areas on T2WI showed that the vessel wall areas of labeled EPC transfusion group were less than those of unlabeled EPC transfusion group and control group fifteen days after artery injury (p,0.05). Histopathological analyses confirmed accumulation and distribution of transfused EPCs at the injury site of LCCA. Conclusions/Significance: These data indicate that MR imaging might be used as an in vivo method for the tracking of EPC

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers

A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of −308 TNF polymorphism function using a novel integrated reporter system

One of the greatest challenges facing genetics is the development of strategies to identify functionally relevant genetic variation. The most common test of function is the reporter gene assay, in which allelic regulatory regions are used to drive the expression of a reporter gene, and differences in expression in a cell line after transient transfection are taken to be a reflection of the polymorphism. Many studies have reported small differences in single nucleotide polymorphism (SNP)-specific reporter activity, including the tumor necrosis factor (TNF) G−308A polymorphism. However, we have established that many variables inherent in the reporter gene approach can account for the reported allelic differences. Variables, such as the amount of DNA used in transfection, the amount of transfection control vector used, the method of transfection, the growth history of the host cells, and the quality and purity of DNA used, all influence TNF −308 SNP-specific transient reporter gene assays and serve as a caution for those researchers who apply this method to the functional assessment of polymorphic promoter sequences. We have developed an integrated reporter system that obviates some of these problems and shows that the TNF G−308A polymorphism is functionally relevant in this improved assay, thus confirming that the −308A allele expresses at a higher level compared with the −308G allele