Preparation of recombinant PTD-HSP27 and verification of its ability to penetrate the cell membrane of human lens epithelial cells and rabbit cornea

AIM: To construct the prokaryotic expression system containing protein transduction domain (PTD) with heat shock protein 27 (HSP27) in order to prepare and purify the recombinant protein, and to verify whether the recombinant protein PTD-HSP27 has the ability to penetrate the human lens epithelial cell (HLEC) membrane and the rabbit cornea. METHODS: The plasmid pKYB-PTD-HSPB1-6His was constructed by the technique of overlap extension PCR. The plasmid was transformed and PTD-HSP27 was purified through nickel affinity chromatography column and identified by Western blotting. PTD-HSP27-6His was labeled with the fluorescein isothiocyanate (FITC). The penetrating ability of PTD-HSP27 into HLECs and rabbit cornea was tested. RESULTS: The recombinant PTD-HSP27 plasmid was successfully cloned and effectively expressed. The correctness of the recombinant protein PTD-HSP27 was demonstrated. Fluorescence microscopic examination showed that PTD-HSP27-FITC was internalized by HLECs. Fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. CONCLUSION: The recombined gene PTD-HSPB1 was constructed by overlap extension PCR technique and the PTD-HSP27 fusion protein was prepared and purified by nickel affinity chromatography column. Using the technique of PTD-fusion protein, HSP27 was transduced into HLECs and passed through the cornea.     

Astragalus polysaccharides protects against free fatty acid-induced human vascular endothelial cell dysfunction via AMPK-eNOS pathway

AIM: To study the protective effect of Astragalus polysaccharides (APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were divided into control group, APS group [APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and compound C group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h]. The cell viability was detected by MTT assay. Nitric oxide (NO) content in the medium was determined by nitrate reductase assay. The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were measured by Western blot. RESULTS: No significant difference of all indexes between APS group and control group was observed. The cell viability in free fatty acid group decreased significantly compared with control group. The cell viability in free fatty acid plus APS group was significantly improved as compared with free fatty acid group. The cell viability in compound C group was almost the same as that in free fatty acid group. The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group. No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed. No significant difference of the AMPK and eNOS protein levels in all groups was found. CONCLUSION: APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.     

Expression of Gab2 in human osteosarcoma U2-OS cells

AIM: To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteosarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells. METHODS: The technique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells. After transfection, through chemotaxis and invasion assays in vitro, the cell migration and invasion abilities were detected. RESULTS: After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells (SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells (Scr/U2-OS) and U2-OS cells. After stimulation with epidermal growth factor (EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells (P<0.01). The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups (P<0.01). CONCLUSION: Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.     

不同基因型抗虫棉的光合生产与叶源特征

以33B为对照,对早发型常规Bt棉鲁棉研17、鲁棉研21,晚发型Bt棉鲁棉研22,杂交Bt棉鲁棉研15、鲁棉研20进行了2年的种植和比较。早发型品种中前期干物质积累和叶面积增长快,但后期干物质积累慢、叶面积下降快,表现出明显的早发早熟和干旱年份易早衰的特点;晚发型品种前期干物质积累和叶面积增长慢,但后期增长快,表现出明显的晚发甚至贪青晚熟的特点。两个杂交棉品种表现出与早发型品种基本相同的生育规律,但中前期叶面积系数和干物质积累量显著高于其它供试品种。盛铃期以前,未见品种间功能叶光合速率的显著差异,但在干旱年份,早发型品种在始絮期功能叶的光合速率显著低于晚发型品种。早发型品种中后期的根冠比和功能叶含K量显著低于晚发型品种,而叶面积载荷量却显著高于晚发型品种。不同品种干物质产量和熟相的差异可能分别与叶面积系数大小和库源比例不同有关。     

棉铃虫对Bt毒蛋白(Cry1 Ac)抗性品系的选育

1 997～2000年,用含Bt毒蛋白(Cry1Ac)的人工饲料对来自本所试验棉田的棉铃虫经过室内21代中16代次的筛选,筛选后F19代LC50值(4.3646g@L1)比筛选前F2代LC50值(0.2972 g@L-1)提高了14.7倍.试验还发现雌性棉铃虫对Bt毒蛋白的敏感性大于雄性.对Bt毒蛋白抗性品系筛选前后分别测定了其不同菌系(商品制剂Dipel和Xentari)的剂量-死亡率回归线,发现Bt毒蛋白抗性品系与其不同菌系间不存在明显的交互抗性.     

大豆吡哆醇生物合成蛋白基因(PDX)的电子克隆和进化分析

电子克隆(In silicocloning)是随着基因组计划和EST计划实施而发展起来的利用生物信息学手段进行基因克隆的新方法。根据物种间同源基因相对保守的特点,以拟南芥(Arabidopsis thaliana)吡哆醇生物合成蛋白cDNA序列为信息探针,对大豆(Glycine max)EST数据库进行同源搜索和序列拼接,获得了1 280 bp长的大豆吡哆醇生物合成蛋白的基因序列(GenBank登陆号为DQ139265)。经过RT-PCR扩增、基因组PCR扩增、分子克隆和序列分析验证,结果表明与电子克隆序列完全一致。该基因具有完整的开放阅读框架(ORF,20~955 bp),编码311个氨基酸。通过与水稻、日本百脉根、烟草、截形苜蓿等物种的吡哆醇生物合成蛋白序列比对,发现该基因具有高度的保守性。表明根据物种间同源基因序列,对跨物种间EST数据库进行同源检索、筛选、拼接,是克隆基因的有效途径。     

淀粉和面筋蛋白对面制品流变特性的影响

面粉的两种主要成分淀粉和蛋白质对面制品的流变特性有显著的影响。概括了影响淀粉糊流变特性的几个主要因素:蒸煮温度、搅拌时间、搅拌速度、pH值以及淀粉糊浓度、盐和蔗糖的作用,同时讨论了面筋蛋白对面制品流变特性的影响机理。     

源库改变对小麦籽粒蛋白质,淀粉含量及产量的影响

利用蛋白质含量及产量各不相同3个春小麦品种．研究了源库改变对淀粉、蛋白质沉术；及单侏产量的影响．结果表明：源库改变明显地影响着籽粒蛋白质的含量，而对淀粉、可溶性糖含量影响不大；品种间蛋白质含量对源库变化的反应不同；千粒重的增减也因品种而异；淀粉的沉积主要受库的限制，而源库都影响着蛋白含量；旗叶在产量和蛋白形成中起着同样重要作用。