Functional studies on three novel HCNH2 mutations in Taiwan: identification of distinct mechanisms of channel defect and dissociation between glycosylation defect and assembly defect

Mutations of the KCNH2 with decreased channel activity lead to congenital long QT syndrome (LQTS). We studied the electrophysiological, glycosylation, trafficking and assembly properties of three novel KCNH2 mutations identified in Taiwanese patients with LQTS (p.N633D, p.R744fs, and p.P923fs). When expressed in HEK293T cells, p.N633D and p.R744fs HERG channels displayed no HERG current while p.P923fs HERG channel showed HERG current with significantly lower (34%) current density and faster inactivation kinetics. In Western blot analysis, pR744fs was the only one with glycosylation defect, which was in consistence with the confocal microscopic findings showing that p.R744fs-GFP was the only one with trafficking defect. However, p.R744fs-GFP differed from pR744fs in being fully glycosylated while p.R744fs fusion with GFP at the N-terminus revealed glycosylation defect. To access the assembly capacity of each mutant, co-immunoprecipitation was conducted. Wild type (WT), p.N633D, and p.P923fs HERG protein showed assembly ability while p.R744fs failed to assemble with neither WT nor itself.In conclusion, we identified three novel LQTS-related KCNH2 mutations and each had a distinct mechanism of channel defect. For p.R744fs mutant, adding GFP to the C-terminus rescued the glycosylation defect but the channel was still assembly defective indicating a dissociation between glycosylation and assembly defects.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Reaction mechanism of isomaltooligosaccharides synthesis by α-glucosidase fromAspergillus carbonarious

Summary An -glucosidase fromAspergillus carbonarious CCRC 30414 was employed for investigating the enzymatic synthesis of isomaltooligosaccharides from maltose. The enzyme transferred a glucose unit from the nonreducing end of maltose and other -linked glucosyl oligosaccharides to glucose and other glucosyl oligosaccharides which function as accepting co-substrates. The transfer of a glucose unit occurs most frequently to the 6-OH position of the nonreducing end of acceptor, but transfer to 4-OH position also occurs. Treatment of 30 % (w/v) maltose with the enzyme under optimum conditions afforded more than 50% isomaltooligosaccharides.     

外来入侵植物银胶菊水提物对三叶鬼针草和茶条木种子萌发的化感作用

以伴生草本植物三叶鬼针草和能源木本植物茶条木为受试植物,初步研究了外来入侵植物银胶菊根、茎、花水浸提液对二者种子萌发的化感效应。根据发芽率、化感效应敏感指数、发芽速率可以看出,银胶菊不同部位的浸提液在不同浓度下对三叶鬼针草的种子萌发的影响力不同;而对茶条木种子萌发无明显的作用。为选择适宜植物,生物替代防治银胶菊提供依据。     

树突状细胞在肾小管间质纤维化中作用及缬沙坦的干预调节

探讨树突状细胞(DC)在肾纤维化大鼠肾小管间质中分布,以及缬沙坦对DC浸润聚集的干预作用。建立肾大部切除大鼠模型,随机分为正常组(n=18),假手术组(n=18),模型组(n=18),缬沙坦治疗组(n=18)。分别于建模1、4、12周取肾组织,采用HE和Masson染色评定各组肾小管间质纤维化(TIF)程度;采用免疫双染及荧光图像分析法,观察DC-SIGN DC在各组大鼠肾组织中分布变化;采用免疫组化方法,观察P-选择素以及TGF-β1、α-平滑肌肌动蛋白(α-SMA)、III型胶元(ColIII)、纤维连接蛋白(FN)在上述肾组织中表达;以及RT-PCR检测P-选择素、TGF-β1、α-SMA、ColIII、FN的mRNA水平。结果显示,(1)模型组DC-SIGN DC主要分布于肾小管、肾间质和肾血管,以肾间质最为明显;其分布数量于12周较1和4周呈明显增多,且与慢性肾功能减退呈正相关。(2)12周时手术组大鼠肾小管间质区P-选择素、TGF-β1、α-SMA、ColIII、FN mRNA转录水平和蛋白质表达均明显增加,并与TIF程度以及DC-SIGN DC分布数量呈正相关。(3)经缬沙坦治疗后,DC-SIGN DC分布减少,以及P-选择素、TGF-β1、α-SMA、ColIII、FN mRNA转录水平和蛋白质表达下降,TIF程度减轻及肾功能改善。研究结果表明,DC启动参与了肾小管间质纤维化形成,并与肾功能损害程度密切相关。缬沙坦对此具有明显的抑制和肾脏保护作用。     

Aspergillus sp. EA-LJS80的分离及其发酵液提取物七叶皂苷C的生物活性

本研究从陕西留坝紫柏山采集的七叶树Aesculus chinensis根和茎中分离出一株具有高产七叶皂苷C的内生真菌,将其编号为EA-LJS80。利用ITS序列分析鉴定其种属并对其形态进行观察;采用紫外-可见分光光度计进行全波长扫描,确定其最大紫外吸收峰;采用HPLC测定七叶皂苷C的产量;采用滤纸片扩散法研究其发酵液提取物的抑菌活性;采用CCK-8法测定提纯后七叶皂苷对肺腺癌A₅₄₉细胞的增殖抑制率,探究其生物活性。结果显示,该菌株EA-LJS80为曲霉菌属Aspergillus真菌,最大紫外吸收波长为230nm,测得提纯后七叶皂苷C的产量为9.23mg/mL。生物活性实验表明：菌株EA-LJS80的发酵液提取物对大肠杆菌Escherichia coli等4种致病菌有明显的抑菌效果;经CCK-8法测定,提纯后的七叶皂苷对肺腺癌A₅₄₉细胞的增殖有明显的抑制作用。综上所述,菌株EA-LJS80具有产七叶皂苷活性,尤其是七叶皂苷C的产量较高,且其产生的七叶皂苷具有较高的生物活性,这对于改进七叶皂苷生产模式,提高生产效率具有重要意义,同时对于七叶皂苷C的进一步研究有一定的促进作用。     

Tea Consumption and Risk of Head and Neck Cancer

Background

The current study evaluated the association between tea consumption and head and neck cancer (HNC) in Taiwan, where tea is a major agricultural product and a popular beverage.

Methods

Interviews regarding tea consumption (frequency, duration, and types) were conducted with 396 HNC cases and 413 controls. Unconditional logistic regression was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) of HNC risk associated with tea drinking, adjusted for sex, age, education, cigarette smoking, betel quid chewing, and alcohol drinking.

Results

A reduced HNC risk associated with tea drinking (OR for every cup per day = 0.96, 95% CI: 0.93–0.99; OR for ≧5 cups per day = 0.60, 95% CI: 0.39–0.94) was observed. The association was especially significant for pharyngeal cancer (OR for every cup per day = 0.93, 95% CI: 0.88–0.98; OR for ≧5 cups per day = 0.32, 95% CI: 0.16–0.66). A significant inverse association between HNC and tea consumption was observed particularly for green tea.

Conclusions

This study suggests that tea drinking may reduce the risk of HNC. The anticancer property of tea, if proven, may offer a natural chemopreventive measure to reduce the occurrence of HNC.