Generation and Evaluation of Recombinant Baculovirus Coexpressing GP5 and M Proteins of Porcine Reproductive and Respiratory Syndrome Virus Type 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of the porcine reproductive and respiratory syndrome, which is one of the most economically devastating diseases of the swine industry. However, whether the inactivated vaccine and modified live attenuated vaccines are effective in disease control is still controversial. Although several groups developed PRRSV virus-like particles (VLPs) as a vaccine against PRRSV, all these VLP-based vaccines targeted PRRSV-2, but not PRRSV-1 or both. Therefore, it is urgent to produce VLPs against PRRSV-1. In this study, we rescued recombinant baculovirus expressing GP5 and M proteins of PRRSV-1 through the Bac-to-Bac® baculovirus expression system. Thereafter, PRRSV VLP was obtained efficiently in the recombinant baculovirus-infected High Five insect cells. Moreover, the PRRSV VLP and PRRSV VLP+A5 could efficiently trigger specific humoral immune responses and B cellular immune responses through intranasal immunization. The combination of PRRSV VLP and A5 adjuvant could improve the level of the immune response. The PRRSV-1 VLPs generated in this study have greater potential for vaccine development to control PRRSV-1 infection.

Chemical constituents from Lonicera japonica flower buds and their anti-hepatoma and anti-HBV activities.

Three new naturally occurring monoterpenoids, japopenoid A (1), japopenoid B (23) japopenoid C (24), and one new caffeoylquinic acid derivative (28), together with thirty-one known compounds (2-22, 25-27, 29-35), were isolated and identified from the flower buds of Lonicera japonica Thunb. Their structures were determined by extensive 1D and 2D NMR spectroscopic methods, high-resolution mass spectrometry, and the absolute configurations of 1, 23, 24 were determined by comparison of their electronic circular dichroism (ECD) spectrum with literature and theoretical calculation. Structurally, compound 1 is a monoterpenoid featured with an unusual tricyclic skeleton. All compounds (1-35) were evaluated for their cytotoxicities against human liver cancer cell lines (HepG 2 and SMMC-7721). Compound 12 exhibited the most potent activity with IC50 values of 26.54 ±â€¯1.95 and 8.72 ±â€¯1.57 µg/ml against HepG 2 and SMMC-7721, and the IC50 values of compound 13 were 26.54 ±â€¯1.95 and 12.35 ±â€¯1.43 µg/ml, respectively. Western blot results further proved that compound 13 induces hepatoma cell apoptosis via the intrinsic apoptosis pathway. In addition, most terpenoids showed inhibitory activity against HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 25 µg/mlof compound 11 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 39.39 ±â€¯5.25, 15.64 ±â€¯1.25, and 16.13 ±â€¯4.10% compared to the control (p < 0.05). These results indicated that L. japonica flower buds could be served as functional food for anti-hepatoma and anti-HBV activities.

Construction, expression and antiviral activity analysis of recombinant adenovirus expressing human IFITM3 in vitro.

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits the replication of multiple pathogenic viruses by blocking their entry. In this study, we constructed a shuttle plasmid, harboring human IFITM3. Thereafter, recombinant adenovirus rAd5-IFITM3 was obtained by co-transfection of the linearized viral backbone vector pAd5 and the shuttle plasmid. The results showed that human IFITM3 did not affect the assembly and morphogenesis of progeny adenovirus. Human IFITM3 can be expressed in both A549 and MDCK cells in a time dependent manner. Furthermore, cells infected with rAd5-IFITM3 at a multiplicity of infection (MOI) of 100 for 24 h were challenged with avian influenza virus (AIV) H5N1 at an MOI of 1 for 6, 12 and 24 h. Rates of H5N1 infection in rAd5-IFITM3 cells were significantly decreased at 24 h post-infection (hpi), in a time dependent manner, compared with that of wild type wtAd5-infected cells. The expressions of viral genes were significantly inhibited at transcriptional and translational levels at 6 and 12 hpi. These results suggest that IFITM3 can suppress H5N1 replication in the early stage of the infection, which may be used as a promise agent against H5N1 infection in vivo.

Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.

[Expression, purification and activity identification of ZtaN-p23 fusion protein in Escherichia coli of Epstein-Barr virus].

AIM: To construct the prokaryotic expression plasmid pGEX-4T-1-BZLF1N-BLRF2, and express it in Escherichia coli. METHODS: The EB virus BZLF1N gene and BLRF2 gene were amplified by RT-PCR respectively. Then, the two genes were linked by splicing overlap extension PCR method and inserted into the vector pGEX-4T-1, and the recombinant plasmid pGEX-4T-1-BZLF1N-BLRF2 was transformed into E.coli BL21 (DE3) strain. The expression protein ZtaN-p23 was analysed by SDS-PAGE and immunoreactivity was proved by Western blotting. RESULTS: Restriction enzyme digestion and DNA sequencing showed recombinant plasmid constructed successfully. The expression product ZtaN-p23 with the molecular weight 46000 was located in the cytoplasm and insoluble. The ZtaN-p23 up to 95% purity was obtained after purified using affinity chromatography. Western blotting showed fusion protein possessed a well bioactivity and specificity. CONCLUSION: The fusion gene BZLF1N-BLRF2 is successfully constructed and effectively expressed in E.coli, which lay the foundation for further research on its biological properties and functions.

Effects of DNMT1 silencing on malignant phenotype and methylated gene expression in cervical cancer cells.

BACKGROUND: DNA methylation has been widely used in classification, early diagnosis, therapy and prediction of metastasis as well as recurrence of cervical cancer. DNMT methyltransferase 1 (DNMT1), which plays a significant role in maintaining DNA methylation status and regulating the expression of tumor suppressor genes. The aim of this research was to investigate the relationship between DNMT1 and abnormal methylation of tumor suppressor genes and malignant phenotype in cervical cancer. METHODS: Levels of DNMT1 mRNA and protein were detected using qPCR and Western blot, respectively. Cell proliferation was analyzed by MTT and apoptosis was performed by Annexin V-FITC/PI double staining flow cytometry, respectively. MeDIP-qPCR and qPCR were performed to measure demethylation status and mRNA re-expression level of 7 tumor-suppressor genes (CCNA1, CHFR, FHIT, PAX1, PTEN, SFRP4, TSLC1) in Hela and Siha cells after silencing DNMT1. RESULTS: The average expression levels of DNMT1 mRNA and protein in Hela and Siha cells were decreased significantly compared with control group. The flow cytometry and MTT results showed that Hela and Siha cells apoptosis rates and cell viabilities were 19.4 ± 2.90%, 25.7 ± 3.92% as well as 86.7 ± 3.12%, 84.16 ± 2.67% respectively 48 h after transfection (P < 0.01). Furthermore, the promoter methylation of five tumor suppressor genes was decreased with the increased mRNA expression after silencing DNMT1, whereas there were no significant changes in PTEN and FHIT genes in Hela cells, and CHFR and FHIT genes in Siha cells. CONCLUSIONS: Our experimental results demonstrate that methylation status of DNMT1 can influence several important tumor suppressor genes activity in cervical tumorigenesis and may have the potential to become an effective target for treatment of cervical cancer.

[Effects of human tissue kallikrein gene delivery on the proliferation of vascular smooth muscle cells].

OBJECTIVE: Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene delivery on the proliferation of vascular smooth muscle cells of SHR (VSMCs(SHR)) induced by platelet derived growth factor-BB (PDGF-BB). METHODS: Primary VSMCs(SHR) were isolated and cultured from thoracic aorta of male SHR. The VSMCs(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazolium (MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27(Kip1) and p21(Cip1). The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCs(SHR). RESULTS: Proliferation of VSMCs(SHR) induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3% (cell counting, n = 3, P < 0.01), 30.2% (MTT, n = 3, P < 0.01) and 36.4% (peak stunning rate of cell-cycle in phase G(0)/G(1)). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27(Kip1) and p21(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n = 3, P < 0.001, respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCs(SHR). CONCLUSION: The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCs(SHR) through Bradykinin B2 receptor and up-regulate expression of p27(Kip1) and p2l(Cip1).

The impacts of ERCC1 gene exon VIII alternative splicing on cisplatin-resistance in ovarian cancer cells.

Excision repair cross complementation group-1 (ERCC1) was reported to be responsible for drug resistance during cancer treatment. In this report, we first proved the existence of ERCC1 exon VIII alternative splicing in ovarian cancer cells. Further investigation showed that over-expressed exon VIII deficient ERCC1 variant failed to change the protein level of ERCC1 in cancer cells, but decreased the excision repair function of ERCC1 and enhanced sensitivity of cancer cells to cisplatin in a dose-dependent manner. The results indicate that ERCC1 exon VIII alternative splicing does exist in some ovarian cancer cell lines, and regulates cisplatin-resistance in ovarian cancer cells.

Confirmation of 42-bp deletion within the ERCC1 5' UTR.

Our previous studies revealed a splicing variant (lacking a 42 base pair segment) within the 5'-UTR of the ERCC1 gene, a critical component of the nucleotide excision repair (NER) pathway that plays an important role in the development of chemoresistance in platinum-based anticancer therapy. This 42-bp segment seems to possess a regulatory function in ERCC1 expression and representing the level of clinical response to platinum-treatment in ovarian cancer patients. To confirm the existence of the 42-bp deletion and to investigate the 42-bp function, we performed several experiments and assays. Northern blot analysis and RNase protection assay provide evidence that the 42-bp deletion occurs at RNA level of ERCC1 5'-UTR in both ovarian cancer cell lines and ovarian cancer tissues. Luciferase assay suggests that this gene fragment possesses a regulatory function as an enhancer of ERCC1 gene expression in ovarian cancer cells. In Electrophoretic Mobility Shift Assay (EMSA), a shift band present in the ovarian cancer cell line extracts is consistent with the presence of an intracellular protein that recognizes this specific 42-bp sequence. Further, specific EMSA results with 42-bp probe mutated at the site of RFX-1 indicate different putative-DNA binding proteins, rather than RFX-1. We conclude that the 42-bp sequence within the 5'-UTR influences the expression of ERCC1 and hence can influence response to cisplatin in ovarian cancer therapy.

Radiotoxicity on bone marrow after 89Sr therapy radiosensitized by nicotinamide and carbogen in mice.

OBJECTIVE: To investigate the radiotoxicity to bone marrow after 89Sr therapy radiosensitized by nicotinamide and carbogen. METHODS: Chinese Kunming, NIH, BALB/c and F1 mice were divided into five groups: negative control (saline), positive control (89Sr), 89Sr+nicotinamide, 89Sr+carbogen and 89Sr+nicotinamide+carbogen. 89SrCl containing activities of 7400 kBq (200 microCi) in 200 microl of saline was administrated by injection into the tail vein. An equal volume of saline only was given to the negative control group. Chinese Kunming and NIH mice were killed on days 1, 2, 3, 4, 6, 8, 15, 20, 30, 60 and 90 after injection. BALB/c and F1 mice were killed on days 60 and 90. Femoral marrow reticulocytes were separated for assay of micronuclei. RESULTS: The average frequency of the reticulocytes is shown in a dual-peak curve after injection. The first maximum frequency occurred between the second and the fourth days, and the second between the tenth and the 14th days. A significant statistical difference in frequency was found between the negative and the positive control groups (P<0.001, F=15.517), while no difference was found among the 89Sr+nicotinamide+carbogen, 89Sr, 89Sr+nicotinamide and 89Sr+carbogen groups (P>0.05, F=0.717) and among the NIH groups, 89Sr, 89Sr+nicotinamide, 89Sr+carbogen and 89Sr+nicotinamide+carbogen (P>0.05, F=1.734). There is also no significant difference in the frequency of reticulocytes between Chinese Kunming, NIH, BALB/c and F1 mice (P>0.05). Although the intervention of the radiosensitizer accelerated the occurrence of micronuclei in reticulocytes, there was no significant statistical difference between the group with radiosensitizer and the groups without it. CONCLUSIONS: The administration of radiosensitizer did not aggravate the toxicity on bone marrow.