The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.     

鸟嘌呤和8-羟基脱氧鸟嘌呤核苷在聚甘氨酸修饰电极上的电化学行为及其同时测定

在优化的实验条件下,利用电化学方法制备了甘氨酸修饰电极,对修饰膜的电活性进行了表征.用循环伏安法研究了鸟嘌呤(G)和8一羟基脱氧鸟嘌呤核苷(8-OH-dG)在聚甘氨酸修饰电极上的电化学行为,并建立了对两者进行分别检测和同时检测的分析方法.实验结果表明,聚甘氨酸修饰电极可以增强鸟嘌呤和8-羟基脱氧鸟嘌呤核苷在电极表面的吸附,并且可以加快鸟嘌呤和8-羟基脱氧鸟嘌呤核苷在电极表面的电子传输,使两种电活性物质在聚甘氨酸修饰电极上的电化学信号明显增大,检测灵敏度大大提高,并且该修饰电极具有良好的稳定性和重现性.可用于鸟嘌呤和8-羟基脱氧鸟嘌呤核昔的分别和同时检测.     

N（2）—乙酰基—9—[（2—乙酰氧乙氧基）甲基]鸟嘌呤的制备工艺条件的研究

人工神经网络由于其具有较好的自组织,自学习,多输入,多输出的能力,在预测方面已取得了广泛应用,本文将BP人工神经网络应用于N（2）－乙酰基－9－[2(2-乙酰氧乙氧基）甲基鸟嘌令的制备工艺条件快速 准确地确定工艺影响参数及其范围的问题。     

阿昔洛韦的合成工艺改进

设计了1条以NaHSO4为催化剂合成阿昔洛韦的新工艺,以醋酐和1,3-二氧戊环为起始原料,合成2-氧杂-1,4-丁二醇二乙酯,然后再与乙酰化后的鸟嘌呤发生缩合制得双乙酰阿昔洛韦,双乙酰阿昔洛韦在Na2CO3水溶液中水解得到目标产物阿昔洛韦。考察了该工艺中反应温度、催化剂用量、溶剂等对反应收率和产品质量的影响。结果表明,在合成关键中间体2-氧杂-1,4-丁二醇二乙酯中,反应温度宜选用60℃;合成双乙酰鸟嘌呤时n（鸟嘌呤）：n（cat）=1.0：0.1为较佳;合成双乙酰阿昔洛韦时选用甲苯作为反应的较佳溶剂。总收率达到79%,并且产物纯度较高,适合工业化生产。     

6-氯鸟嘌呤合成工艺的改进

以鸟嘌呤为起始原料,首先鸟嘌呤与N,N-二甲基甲酰胺和三氯氧磷反应,其后调节溶液pH值在3-4范围内进行水解反应,制得2-甲酰氨基-6-氯嘌呤,再经碱水解、中和得6-氯鸟嘌呤。考察了投料方式、重结晶溶剂对收率的影响,得到适宜的工艺条件为：投料时,先将三氯氧磷在低温下滴入N,N-二甲基甲酰胺配成混合溶液,其后滴入鸟嘌呤,在1,2-二氯乙烷中进行反应;重结晶溶剂选用N,N-二甲基甲酰胺,产品收率达78%（以鸟嘌呤计）,纯度为99%。     

Influence of Different Salts on the G-Quadruplex Structure Formed from the Reversed Human Telomeric DNA Sequence

G-rich telomeric DNA plays a major role in the stabilization of chromosomes and can fold into a plethora of different G-quadruplex structures in the presence of mono- and divalent cations. The reversed human telomeric DNA sequence (5′-(GGG ATT)₄; RevHumTel) was previously shown to have interesting properties that can be exploited for chemical sensing and as a chemical switch in DNA nanotechnology. Here, we analyze the specific G-quadruplex structures formed by RevHumTel in the presence of K⁺, Na⁺, Mg²⁺ and Ca²⁺ cations using circular dichroism spectroscopy (CDS) and Förster resonance energy transfer (FRET) based on fluorescence lifetimes. CDS is able to reveal strand and loop orientations, whereas FRET gives information about the distances between the 5′-end and the 3′-end, and also, the number of G-quadruplex species formed. Based on this combined information we derived specific G-quadruplex structures formed from RevHumTel, i.e., a chair-type and a hybrid-type G-quadruplex structure formed in presence of K⁺, whereas Na⁺ induces the formation of up to three different G-quadruplexes (a basket-type, a propeller-type and a hybrid-type structure). In the presence of Mg²⁺ and Ca²⁺ two different parallel G-quadruplexes are formed (one of which is a propeller-type structure). This study will support the fundamental understanding of the G-quadruplex formation in different environments and a rational design of G-quadruplex-based applications in sensing and nanotechnology.     

Electron Holes in G-Quadruplexes: The Role of Adenine Ending Groups

The study deals with four-stranded DNA structures (G-Quadruplexes), known to undergo ionization upon direct absorption of low-energy UV photons. Combining quantum chemistry calculations and time-resolved absorption spectroscopy with 266 nm excitation, it focuses on the electron holes generated in tetramolecular systems with adenine groups at the ends. Our computations show that the electron hole is placed in a single guanine site, whose location depends on the position of the adenines at the 3′ or 5′ ends. This position also affects significantly the electronic absorption spectrum of (G⁺)^● radical cations. Their decay is highly anisotropic, composed of a fast process (<2 µs), followed by a slower one occurring in ~20 µs. On the one hand, they undergo deprotonation to (G-H2)^● radicals and, on the other, they give rise to a reaction product absorbing in the 300–500 nm spectral domain.     

酿酒酵母和大肠杆菌鸟嘌呤脱氨酶基因的克隆、原核表达及活性测定

与植物体内合成路径不同,微生物体内合成咖啡碱存在一条以黄嘌呤为底物,利用鸟嘌呤脱氨酶催化鸟嘌呤生成黄嘌呤有效合成咖啡碱的新途径。为克隆鸟嘌呤脱氨酶的基因,构建可高效合成黄嘌呤的原核表达载体并对外源蛋白活性进行检测,分别以酿酒酵母和大肠杆菌为研究材料,根据GenBank中酿酒酵母和大肠杆菌中鸟嘌呤脱氨酶基因gud1和egud序列设计引物,聚合酶链式反应特异扩增其基因片段,将目的基因连接至pMAL-c5X载体,转入大肠杆菌BL21（DE3）中诱导蛋白表达,并用高效液相色谱法鉴定其目的蛋白的催化活性。结果表明重组载体pMAL-gud1、pMAL-egud均可用来合成黄嘌呤,且GUD1比EGUD合成黄嘌呤的效率更高。研究结果将进一步丰富黑茶加工技术理论,同时为体外构建高效咖啡碱生物工程菌提供理论支持。