Voltage-Dependent Anion Channel-1, a Possible Ligand of Plasminogen Kringle 5.

Kringle 5, the fifth fragment of plasminogen, is known to be important for inhibiting the proliferation and migration of vascular endothelial cell (VEC), while not having any effects on normal endothelial cells. Therefore, it may be a potential tumor therapy candidate. However, the ligand of the Kringle 5 in VEC has not yet been identified. In this study, the possible ligand of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance (SPR). After four rounds of panning, the specific clones of Kringle 5 were confirmed using enzyme-linked immunosorbent assay (ELISA). The gene sequence analysis showed that they expressed the common amino sequence IGNSNTL. Then, using a NCBI BLAST, 103 matching sequences were found. Following the molecular docking evaluation and considering the acting function and pathway of the plasminogen Kringle 5 in the human body, the most promising candidate was determined to be voltage-dependent anion channel-1 (VDAC-1), which was able to bind to Kringle 5 at -822.65 J·mol-1 of the binding energy at the residues of Lys12, Thr19, Ser57, Thr188, Arg139, Asn214, Ser240 and Lys274. A strong dose-dependent interaction occurred between the VDAC-1 and Kringle 5 (binding constant 2.43 × 103 L·mol-1) in SPR observation. Therefore, this study proposed that VDAC-1 was a potential ligand of plasminogen Kringle 5, and also demonstrated that the screening and validation of protein ligand using phage display peptide library with the molecular docking, along with SPR, was a practicable application.

PTEN-mRNA engineered mesenchymal stem cell-mediated cytotoxic effects on U251 glioma cells.

Mesenchymal stem cells (MSCs) have been considered to have potential as ideal carriers for the delivery of anticancer agents since the capacity for tumor-oriented migration and integration was identified. In contrast to DNA-based vectors, mRNA synthesized in vitro may be readily transfected and is mutagenesis-free. The present study was performed in order to investigate the effects of phosphatase and tensin homolog (PTEN) mRNA-engineered MSCs on human glioma U251 cells under indirect co-culture conditions. PTEN-bearing mRNA was generated by in vitro transcription and was transfected into MSCs. The expression of PTEN in transfected MSCs was detected by immunoblotting, and the migration ability of MSCs following PTEN-bearing mRNA transfection was verified using Transwell co-cultures. The indirect co-culture was used to determine the effects of PTEN-engineered MSCs on the viability of U251 glioma cells by luminescence and fluorescence microscopy. The synthesized PTEN mRNA was expressed in MSCs, and the expression was highest at 24 h subsequent to transfection. An enhanced migration rate was observed in MSCs transfected with PTEN mRNA compared with non-transfected MSCs (P<0.05). A significant inhibition of U251 cells was observed when the cells were cultured with conditioned medium from PTEN mRNA-engineered MSCs (P<0.05). The results suggested that anticancer gene-bearing mRNA synthesized in vitro is capable of being applied to a MSC-mediated anticancer strategy for the treatment of glioblastoma patients.

Cloning, expression and identification of an isoform of human stromal cell derived factor-1α.

Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1ß, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with the recombinant plasmid pCMV-SPORT6-hSDF1 as the template, and the prokaryotic expression vector pET15b-hSDF-1α was constructed. This hSDF-1α was successfully expressed as an inclusion body in Escherichia coli BL21(DE3). The recombinant hSDF-1α was refolded in vitro and separated by cation exchange chromatography. Following these two steps the purity of the hSDF-1α was able to reach >85%. The recombinant hSDF-1α was then purified by size-exclusion chromatography. SDS-PAGE analysis demonstrated that the purity of the hSDF-1α was >95%, which meets almost all the requirements of a protein experiment. Chemotactic activity of the recombinant hSDF-1α was analyzed by Transwell migration assay and it was found that the recombinant hSDF-1α was able to stimulate THP-1 cell migration. These data suggest that the procedure of producing recombinant hSDF-1α proteins with chemotactic activity was feasible and the N-terminal signal peptide of hSDF-1α has little effect on the chemotactic activity of hSDF-1α.

PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for ß-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel ß-cell source for the treatment of diabetes.

Two-step chromatographic method for separation and purification of nerve growth factor from venom of Chinese cobra.

By selecting the different combination schemes, a simple, fast and highly efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese cobra is reported in this paper. This purification process consists of a two-step chromatographic separation on DEAE-Sepharose F.F. anion-exchange medium followed by a Sephadex G-50 gel filtration. On reducing and non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the nerve growth factor obtained with this process proved to be homogeneous and its molecular weight was separately estimated to be approximately 14.5 and 29.0kD, which was consistent with that reported in literature; and on high performance size-exclusion chromatography and reversed-phase chromatography, its purity was about 99%. The yield of this purification method was 0.51% and the nerve growth factor obtained had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. The optimum concentration of nerve growth factor was 5-100ng/ml and the minimal concentration eliciting neurite outgrowth from chick embryonic dorsal root ganglia was 5.0 ng/ml.