[Effect of overexpression of transcription factor Runx2 and Osterix on osteogenic differentiation of endothelial cells].

PURPOSE: To explore the effect of overexpression of Runx2 and Osterix (OSX) genes on osteogenic differentiation of human umbilical vein endothelial cells (HUVECs). METHODS: Overexpressed Runx2 and OSX lentiviral vectors were transfected into HUVECs respectively. The osteogenic potential of transfected cells was identified by alkaline phosphatase (ALP) staining and ALP activity. Furthermore, real time-PCR, Western blot and immunofluorescence staining were performed to detect the expression of osteogenic genes and proteins in HUVECs. GraphPad Prism 6.01 software was used for statistical analysis. RESULTS: Overexpression of Runx2 gene was beneficial for osteogenic differentiation of HUVECs, while overexpression of osterix gene did not show osteogenic differential potential. Moreover, overexpression of Runx2 gene in HUVECs up-regulated the gene expression level of Runx2, OSX, ALP, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), and up-regulated protein level of OPN and OCN. CONCLUTIONS: Overexpression of Runx2 could promote osteogenic differentiation of HUVECs.

Electrochemical scanning tunneling microscope imaging of self-assembled monolayer of double-stranded DNA on Au(111).

Self-assembled monolayer of a double-stranded DNA, which was formed by a 20-nt 5'-thiol-modified oligonucleotide at the interface of Au(111)/solution, has been imaged and studied with electrochemical scanning tunneling microscopy (ECSTM). The results showed that the preferred directions of the adsorbed DNA were <110> and <112>. The measured width of the DNA stripes was 0.95 +/- 0.02 nm, which was consistent with the width of dsDNA base pairs. The contour of DNA in the STM image consisted of a series of blobs, which were due to the anchoring of the bases to the substrate along the dsDNA chain. The imaging of a large-scale array of DNA lying flat on the substrate offers a convenient method for the further application of high-resolution STM to the study of the interaction between DNA and a variety of molecules.

Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra.

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.

cDNA cloning and characterization of Agkistin, a new metalloproteinase from Agkistrodon halys.

Agkistin was a new snake venom metalloproteinase (SVMP) gene which was cloned from Agkistrodon halys. Its deduced amino acid sequence has two additional cysteines (Cys407 and Cys426) in the disintegrin domain compared to other RGD containing SVMPs. The full-length gene (Agkistin) and its disintegrin region (named Agkistin-s) were expressed by baculovirus expression system (pFastBac-Htb vector) with His-tag, and their platelet aggregation-inhibition activity was evaluated. The expressed protein Agkistin can also induce apoptosis of HMEC cells in the basal medium after incubated at 37 degrees C for 20 h.