With the younger onset of diabetes, the reproductive dysfunction caused by diabetes has received widespread attention. Hyperglycemia and insulin resistance, the main features of diabetes, are high-risk factors for male reproductive dysfunction. Obesity, the common co-morbidity of diabetes, may aggravate the progression of reproductive dysfunction. Glucagon-like peptide-1(GLP-1) and its receptor agonists improve the overall health status by lowering blood glucose, reducing body weight, and inhibiting inflammatory response, which indirectly exerts protective effects on the reproductive system. GLP-1 also protects reproductive function by regulating the neuroendocrine function, and directly acting on the supporting cells and interstitial cells of the testis. However, some studies did not find the protective effects. High-quality clinical studies are needed. GLP-1 receptor agonists may be a therapeutic option to improve reproductive dysfunction in diabetic men.

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P < 0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.
Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Humans ; Tumor Suppressor Protein p53 ; pharmacology

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
Cell-Penetrating Peptides ; biosynthesis ; genetics ; pharmacology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Objective To clone the full length staphylococcal protein A(SPA) gene from Staphylococcus aureus (ATCC6538), and subsequently study the gene structure and antibody binding ability.Methods The full length and the functional region of the SPA gene were cloned into pHisSUMO vector respectively, and expressed in E. coli. The full length and the functional fragment of the SPA protein were detected for antibody binding ability and stability. The functional fragment of the SPA protein fused with SUMO was coupled to the CNBr-activated agarose for antibody purification from rabbit serum. Results A variant of the full length SPA gene was cloned, which has been submitted to GenBank (the accession number is EU695225). Two fusion proteins had the same antibody binding ability as the untagged SPA protein. However, the formers was more stable than the latter at the tested conditions. SUMO-SPA conjugated-agarose kept high efficiency for antibody binding. Conclusion To our knowledge, the full length SPA gene of S.aureus(ATCC6538) is a novel variant. The SUMO tag can improve the stability of the functional region of the SPA protein without damaging the antibody binding ability. This fusion protein has been used for antibody purification successfully.

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa-621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375 x 10(4) U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.
Cloning, Molecular ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; enzymology ; Solubility