The peripheral nervous system has the potential for full regeneration following injury and recovery, predominantly controlled by Schwann cells (SCs). Therefore, obtaining a sufficient number of SCs in a short duration is crucial. In the present study, rat adipose-derived stem cells (ADSCs) were isolated and cultured, following which characterization of the ADSCs was performed using flow cytometry. The results showed that the cells were positive for the CD29 and CD44 markers, and negative for the CD31, CD45, CD49 and CD106 markers. The multilineage differentiation potential of the ADSCs was assayed by determining the ability of the cells to differentiate into osteoblasts and adipocytes. Following this, the ADSCs were treated with a specific medium and differentiated into Schwann-like cells. Immunofluorescence, western blot and reverse transcription-quantitative polymerase chain reaction analyses showed that ~95% of the differentiated cells expressed glial fibrillary acidic protein, S100 and p75. In addition, the present study found that a substantial number of SCs can be produced in a short duration via the mitotic feature of Schwann-like cells. These data indicated that Schwann-like cells derived from ADSCs can undergo mitotic proliferation, which may be beneficial for the treatment of peripheral nerve injury in the future.

To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.

Girdin, an actin-binding protein, is associated with cell migration and is expressed at high levels in glioma cells. However, the association between girdin and the development of glioma remains to be elucidated. In the present study, short-hairpin RNA technology was used to silence the gene expression of girdin. The effects of girdin silencing on glioma cell proliferation, migration and invasion were then assessed using a cell viability assay, wound-healing assay, transwell invasion assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis and gelatin zymography. The results suggested that girdin silencing inhibited the proliferation, migration and invasion of glioma cells. In addition, the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9 were also affected by girdin silencing. Further mechanistic investigation indicated that girdin may regulate glioma cell migration and invasion through the phosphatidylinositol-3-kinase/protein kinase B (PI3K-Akt) signaling pathway. Therefore, the results of the present study provide a theoretical foundation for the development of anticancer drugs.

The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2′-O-methyl nucleotide (2′-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2′-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.