Optimization of sulfated modification conditions of tremella polysaccharide and effects of modifiers on cellular infectivity of NDV

Based on our previous research, sulfated modification conditions of Tremella polysaccharide (TPS), the chlorosulfonic acid to pyridine (CSA-Pry) ratio, reaction temperature and time, were optimized by L₉ (3⁴) orthogonal design taking the yield and degree of sulfation (DS) of modifiers as indexes. Two TPSs, TPS_tp and TPS_70c, were modified under optimized conditions. The effects of two modifiers, sTPS_tp and sTPS_70c, on cellular infectivity of NDV were determined by MTT method taking the non-modified TPS_tp, TPS_tc and TPS_70c as controls. The results showed that the optimized modification conditions were reaction temperature of 80 °C, CSA-Pry ratio of 1:6 and reaction time of 1.5 h. Five polysaccharides at proper concentrations could significantly inhibit the infectivity of NDV to CEF. The virus inhibitory rates of sTPS_tp at 1.563 μg mL⁻¹ group were the highest and significantly higher than those of other three non-modified polysaccharide groups in three sample-adding modes. This indicated that sulfated modification could significantly improve the antiviral activity of TPS. sTPS_tp possessed the best efficacy and would be as a component of antiviral polysaccharide drug.     

Soluble expression, purification and functional identification of a disulfide-rich conotoxin derived from Conus litteratus

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6 mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel microO-conotoxin.     

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

Nuclear retention of the lncRNA SNHG1 by doxorubicin attenuates hnRNPC–p53 protein interactions

The protein p53 plays a crucial role in the regulation of cellular responses to diverse stresses. Thus, a major priority in cell biology is to define the mechanisms that regulate p53 activity in response to stresses or maintain it at basal levels under normal conditions. Moreover, further investigation is required to establish whether RNA participates in regulating p53's interaction with other proteins. Here, by conducting systematic experiments, we discovered a p53 interactor—hnRNPC—that directly binds to p53, destabilizes it, and prevents its activation under normal conditions. Upon doxorubicin treatment, the lncRNA SNHG1 is retained in the nucleus through its binding with nucleolin and it competes with p53 for hnRNPC binding, which upregulates p53 levels and promotes p53‐dependent apoptosis by impairing hnRNPC regulation of p53 activity. Our results indicate that a balance between lncRNA SNHG1 and hnRNPC regulates p53 activity and p53‐dependent apoptosis upon doxorubicin treatment, and further indicate that a change in lncRNA subcellular localization under specific circumstances is biologically significant.     

Targeting of focal adhesion kinase by small interfering RNAs reduces chondrocyte redifferentiation capacity in alginate beads culture with type II collagen

Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell–extracellular matrix (ECM) interactions, and chondrocyte–type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte–type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte‐specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p‐FAK_Y397, and p‐ERK1/2. In contrast, expression levels of p‐FAK_Y397 and p‐ERK1/2, but not p‐Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF‐β1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK‐siRNA) inhibited mRNA expression of type II collagen and SOX‐6 compared to the control. These FAK‐siRNA‐transfected cells could not recover type II collagen even in the presence of TGF‐β1 or type II collagen in alginate beads culture. We also found that FAK‐siRNA‐transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation. J. Cell. Physiol. 218: 623–630, 2009. © 2008 Wiley‐Liss, Inc.     

改性聚二甲基硅氧烷 (iPDMS) 蛋白芯片在肿瘤相关抗原自身抗体筛查中的应用

肿瘤标记的快速筛查是临床早期诊断的难题。利用化学发光蛋白质芯片技术,对低丰度的肿瘤相关抗原的自身抗体进行高灵敏度的筛选,是一种有益的尝试。本研究首先将带烯烃末端的、引发聚合反应的表面引发剂加入到常规聚二甲基硅氧烷材料中,再通过热交联反应固定到聚二甲基硅氧烷的三维结构中,形成改性聚二甲基硅氧烷 (iPDMS)。为了使iPDMS材料具有抗蛋白质非特异性吸附的特性,在活性引发位点处通过表面引发的原子转移自由基聚合反应合成poly(OEGMA) 高分子刷。最后将20种肿瘤相关的抗原利用高通量喷点打印技术打印到芯片的特定区域,并组装成iPDMS芯片的48孔检测微孔板。对临床上常见的8种肿瘤患者血清进行分析,发现VEGFR1和VEGF121自身抗体对常见的8种肿瘤具有检测价值,有望成为肿瘤快速筛查的检测指标。     

酱香型白酒酿造酒醅中酵母菌多样性研究

为解析酱香型白酒酿造酒醅中酵母菌的菌群结构,获取酒醅中的主要酵母菌,采用高通量测序法分析酱香型白酒酒醅中酵母菌多样性及主要功能菌群,同时采用可培养分离方法获取酒醅中酵母菌活性菌株。从酱香型白酒下沙至五轮次酒醅中共检出59个属、129个种的酵母菌,分离得到酵母菌活性菌株41种,检测到的酵母菌种类与获得的酵母菌活菌在各香型白酒中最多。不同时期酒醅中的酵母菌种类和数量差异明显,其中下沙、造沙轮次以Pichia kudriavzevii为绝对优势酵母菌;一至五轮次随着轮次的递增,酒醅中优势酵母菌的种类增多,其中主要的优势酵母菌有Pichia kudriavzevii、Pichia manshurica、Zygosaccharomyces bailii、Saccharomyces cerevisiae、Candida apicola。酱香型白酒酒醅中蕴藏着极其丰富的酵母菌资源,对酵母菌菌群结构的解析有助于科学地认识酱香型白酒酿造过程中产酒与风味代谢机理,为发酵过程的调控提供一定依据。     

植物LEAFY 同源基因的研究进展

本文就近10年来LEAFY(简写为LFY)同源基因的研究进展做了综合分析。通过对19种植物中已分离到的LFY同源基因的序列比较分析发现: LFY同源基因编码区核苷酸和氨基酸序列同源性都较高;在双子叶植物基因组中, 拷贝数却有所不同。该基因的表达特性显示其在不同植物中表达的时间和空间有所差异。根据已知序列推导的氨基酸序列构建的系统进化树表明, 单子叶植物与裸子植物的亲缘关系近于双子叶与裸子植物的亲缘关系。上述研究资料为植物成花机理研究提供了重要参考, 且在研究植物系统进化方面也具有重要的意义。