Genome-wide identification, phylogeny, and expression analysis of Sec14-like PITP gene family in sugarcane.

KEY MESSAGE: Six Sec14-like PITP genes from sugarcane were identified, two of them were cloned, and their biological functions were characterized indicating their involvement in plant defense against biotic and abiotic stresses. Sec14, a phosphatidylinositol transfer protein (PITP) is widely present in eukaryotes. In this study, the structure and expression patterns of six Sec14-like PITP genes (ScSEC14-1, ScSEC14p, ScSFH1, ScSFH2, ScPATL1, and ScPATL2) from sugarcane were analyzed, and two of them (ScSEC14-1 and ScSEC14p) were cloned and functionally verified. Phylogenetic analysis divided these genes into four groups, including group I (ScSFH1 and ScSFH2), group II (ScPATL1 and ScPATL2), Group III (ScSEC14p), and group V (ScSEC14-1). qRT-PCR analysis showed tissue-specific expression of these genes, primarily in the root, leaf, and bud tissues. They responded differently to SA, MeJA, and ABA stresses. ScSEC14-1, ScSEC14p, and ScSFH2 were upregulated by CuCl2 and CdCl2, while ScSEC14-1, ScSFH1, ScSFH2, and ScPATL1 were upregulated by PEG and NaCl. When infected by Sporisorium scitamineum, the transcripts of ScSFH1, ScSFH2, ScPATL1, and ScPATL2 were upregulated in the resistant genotype Yacheng 05-179, while those of ScSEC14-1 and ScSEC14p were upregulated in the susceptible genotype ROC22. Subcellular localization showed that ScSEC14-1 and ScSEC14p were mainly localized in the plasma membrane and cytoplasm. Enhanced growth of Escherichia coli BL21 cells expressing ScSEC14-1 and ScSEC14p showed high tolerance to NaCl and mannitol stresses. The transient overexpression of ScSEC14-1 and ScSEC14p in Nicotiana benthamiana leaves enhanced its resistance to the infection of tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. We can conclude the involvement of ScSEC14-1 and ScSEC14p in the defense against biotic and abiotic stresses, which should facilitate further research on Sec14-like PITP gene family, especially its regulatory mechanisms in sugarcane.

Effect of silencing AEG-1 with small interfering RNA on the proliferation and cell cycle of gastric carcinoma SGC-7901 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the effect of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell proliferation and cell cycle of gastric carcinoma cells, and its possible molecular mechanism.</p><p><b>METHODS</b>Control siRNA and AEG-1 siRNA were transfected into gastric carcinoma SGC-7901 cells. 48 h after transfection, the cells were divided into 3 groups including untransfected, siRNA control and AEG-1 siRNA transfection groups. Expressions of AEG-1 mRNA and protein in the 3 group cells were detected by real-time quantitative PCR and Western blot. The changes of cell proliferation were examined using CCK-8 kit, and the cell cycle distribution was detected by flow cytometry. Finally, expressions of cell proliferation and cell cycle related proteins were detected by Western blot.</p><p><b>RESULTS</b>Real-time quantitative PCR and Western blot demonstrated that compared with the untransfected and siRNA control groups, expressions of AEG-1 mRNA and protein were significantly down-regulated in the AEG-1 siRNA transfection group (P < 0.05), but there was no significant difference between the untransfected and siRNA control groups (P > 0.05). Furthermore, in vivo experiment confirmed a significant down-regulation of AEG-1 protein in the AEG-1 siRNA transfection group (P < 0.05). In addition, AEG-1 siRNA obviously inhibited the proliferation of SGC-7901 cells at different time points after transfection with AEG-1 siRNA. The percentage of cells in G0/G1 phase in the AEG-1 siRNA transfection group [(61.26 ± 1.25)%] was significantly higher than those in the untransfected group [(46.17 ± 1.91)%] and siRNA control group [(46.46 ± 1.96)%], and there was a significant difference between them (all P < 0.001). Furthermore, the result of Western blotting revealed that down-regulation of AEG-1 expression evoked the down-regulation of cdk2 and cyclin D1 expressions and elevation of p21 expression in vitro and in vivo.</p><p><b>CONCLUSIONS</b>The inhibition of cell proliferation and cell cycle arrest mediated by down-regulation of AEG-1 expression may be closely associated with the changes of expression of cell cycle related proteins including cdk2, cyclin D1 and p21.</p>

Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease.</p><p><b>METHODS</b>The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis.</p><p><b>RESULTS</b>We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system.</p><p><b>CONCLUSION</b>The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.</p>

Influence of dendritic cell infiltration on prognosis and biologic characteristics of progressing gastric cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the relation between dendritic cell (DC) infiltration and clinicopathologic parameters, biologic characteristics and prognosis of progressing gastric cancer.</p><p><b>METHODS</b>The development of apoptotic cell death (apoptotic index, AI) in 61 progressing gastric carcinoma tissues was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. The PCNA labeling index (PCNA-LI), density of dendritic cells in the tumor were detected by immunohistochemical method by the LSAB kit using antibody against S-100 protein and PC-10.</p><p><b>RESULTS</b>DC infiltration was negatively correlated with lymph node metastasis, clinical stage and PCNA-LI, but positively with AI. The DCs in gastric cancer groups with and without lymph node metastasis were (5.63 +/- 4.37)/HPF and (8.51 +/- 5.57)/HPF with difference significant (P < 0.05). The DC infiltration in I, II, III stage lesions were (11.23 +/- 6.05)/HPF, (6.28 +/- 4.37)/HPF and (5.53 +/- 5.19)/HPF also with differences significant (P < 0.01). The PCNA-LI was significantly higher in the low DC group (57.10% +/- 14.18%) than that of high DC group (48.15% +/- 10.59%, P < 0.01). AI findings were 3.77% +/- 1.26% and 2.95% +/- 1.07% in the high and low DC groups (P < 0.01). A positive correlation was observed between DC infiltration and AI (r = 0.39, P < 0.01) whereas a negative correlation between DC infiltration and PCNA-LI (r = -0.47, P < 0.01). The prognosis of high DC infiltration patients was significantly better than those with low ones.</p><p><b>CONCLUSION</b>The infiltrating dendritic cells in and around tumor, representing the local immune status of the host, may play an important role in immunological defense mechanism of host versus tumor. Dendritic cells may inhibit the proliferation and induce the apoptosis of the tumor cells, thus affecting the clinical features and improve the prognosis of gastric carcinoma.</p>