Modulation of the basal activity of N-acetylglucosaminyltransferase V by phosphatidylinositol-3-kinase/protein kinase B signaling pathway in human hepatocarcinoma cells

The modulation of GnT-V activity by signaling molecules in PI-3-K/PKB pathway in human hepatocarcinoma cell line 7721 was studied. GnT-V activity was determined after the transfection of sense or antisense cDNA of PKB into the cells, as well as the addition of activators, specific inhibitors, and the antibodies to the enzyme assay system or culture medium. It was found that the basal activity of GnT-V was up regulated by the sense and down regulated by the antisense cDNA of PKB transfected into 7721 cells. GnT-V was activated by PIP₂, PIP₃ or GTP[S] added to the assay system, and the activation of PIP₂ or GTP[S] was abolished by LY2940002, a specific inhibitor of PI-3-K, but the activation of PIP₃ was not attenuated by LY2940002. In addition, GnT-V activity in cultured parental or H-ras transfected cells was inhibited by the antibody against PKB or PI-3-K. These findings demonstrated the involvement of PI-3-K/PKB signaling pathway in the regulation of GnT-V. Moreover, ET18-OCH₃, an inhibitor of Raf translocation and PI-PLC enzyme, which produces the activator of PKC, as well as the antibodies against Raf-1 or MEK also inhibited GnT-V activity in the parental and H-ras transfected cells. The inhibitory rates, however, were less in the transfected cells than those in the parental cells. These results reveal that in parental and H-ras transfected 7721 cells, the basal activity of GnT-V is also regulated by the Ras/Raf-1/MEK/MAPK cascade in addition to PI-3-K/PKB signaling pathway. The significance of these two pathways in the regulation of GnT-V and their relations to the activation of PKC previously reported by our laboratory (Ju TZ et al., 1995 Glyconjugate J 12, 767–772) was discussed.     

水体热分层对万峰湖水环境的影响

2009年9月(秋季)和2010年1月(冬季)对南盘江流域峡谷型水库--万峰湖的水温和水化学(DO、pH、总磷)进行监测,结果表明:万峰湖水体在秋、冬季节均存在明显分层,秋季.水体分为3层,0～10 m为混匀层,10～50 m为斜温层,50 m以下为滞水层,这种温度分层阻止了水的对流混合,引起显著的水化学分层,形成底部厌氧层.冬季,水温下降,在水下50 m左右分层,表层随深度增加水温下降,下层为滞水层,水温较为均匀.相关性分析表明,万峰湖在2009年9月(秋季)和2010年1月(冬季)水温和DO、pH、总磷之间均为极显著相关,水化学分层与温度分层同步.     

基于Renyi熵滤波的光声图像重建算法设计与实现

针对光声图像重建过程中存在的原始光声信号信噪比差、重建图像对比度低、分辨率不足等问题,提出了基于Renyi熵的光声图像重建滤波算法.该算法首先根据原始光声信号的Renyi熵分布情况,确定分割阈值,并滤除杂波信号;再利用滤波后的光声数据进行延时叠加光声图像重建.利用该滤波算法分别处理铅笔芯横截面(零维)、头发丝(一维)以及小鼠大脑皮层血管(二维)等不同维度样本的光声信号,实验结果表明:相比Renyi熵处理之前,重建图像对比度平均增强了32.45%,分辨率平均提高了30.78%,信噪比提高了47.66%,均方误差降低了35.01%;相比典型的滤波处理算法(模极大值法和阈值去噪法),本研究中图像的对比度、分辨率和信噪比分别提高了25.94%/10.60%、27.90%/19.48%、35.21%/10.60%,均方误差减小了28.57%/16.66%.因此,选择利用Renyi熵滤波算法处理光声信号,从而使光声图像重建质量得到大幅改善.     

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.     

Relations of the type and branch of surface N-glycans to cell adhesion,migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi -mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [³H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin 51 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated 5, but not 1, and antisense GnT-V decreased both 5 and 1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.     

贵州红枫湖水体叶绿素a的分布与磷循环

于2009年8月(夏季)和2010年1月(冬季)在贵州红枫湖采集了分层湖水和分层沉积物样品,分析了湖水样品的总N(TN)、总P(TP)及叶绿素a(Chl-a)含量,结果表明,湖水TN含量在2个季节无明显变化,平均含量为1.58±0.73 mg·L-1,湖水TP含量夏季(0.091±0.070 mg·L-1)高于冬季(0.026±0.055 mg·L-1).夏季湖水在8 m处有季节性分层,下层湖水TN、TP含量高于上层;夏季湖水Chl-a主要集中在上层,上层平均含量为33.2±13.0 mg·m-3,冬季湖水Chl-a平均含量为11.1±3.7 mg·m-3,分析发现,湖水上层(8 m)Chl-a与TP有明显的线性相关关系(r=0.965,P＜0.01),表明红枫湖富营养化主要受P元素限制.沉积物孔隙水中的溶解态P(DP)浓度和湖水的磷酸盐(PO3-4-P)浓度比上覆水体高,具有向上扩散的趋势,利用费克第一定律计算了沉积物向上覆水体的释P速率,发现夏季沉积物向上覆水体释P速率高于冬季,可能主要是由于夏季湖水底层的还原环境下沉积物表层的早期成岩作用生成磷酸盐进入孔隙水而促进了沉积物向上覆水体释放P.根据通量释放结果估算了全湖沉积物向水体的释P通量,约为每年5.0±5.6 t.红枫湖富营养化受P控制,沉积物向水体有很大的释放P的潜力,是湖水P的重要内源,严格控制流域的外源输入才能彻底治理该湖的富营养化.     

Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.     

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.