Structure-Guided Design of Novel l-Cysteine Derivatives as Potent KSP Inhibitors

l-cysteine derivatives as selective KSP inhibitors. Here, we report further optimizations using docking modeling in the L5 allosteric binding site, which led to the discovery of several high affinity derivatives with two fused phenyl rings in the trityl group giving low nanomolar range KSP ATPase inhibition. The representative derivatives potently inhibited cell growth of HCT116 cells in correlation with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.     

Competitive inhibition of phosphoinositide hydrolysis by the muscarinic receptor antagonist AF-DX 116 is of low affinity in mouse cerebral cortex

Carbamylcholine stimulated [³H]inositol phosphate accumulation in mouse cerebral cortical slices with an ED₅₀ value of approximately 70 μM. Increasing concentrations of the M₂ selective muscarinic cholinergic receptor antagonist, AF-DX 116 (0.3–3.0 μM), produced parallel shifts to the right for concentration-response curves to carbamylcholine. A pA₂ value for AF-DX 116 of 6.5 (low affinity) was obtained frommSchild plot analysis. It is concluded that the M₂ muscarinic receptor subtype, as defined by high affinity [³H]AF-DX 116 radioligand binding, is not appreciably coupled to polyphosphoinositide hydrolysis in the mouse cerebral cortex.     

Stat3靶向RNA干扰重组体的构建及鉴定

目的设计针对Stat3的发夹样RNA干扰重组体，通过脂质体转染结肠癌细胞HCT116观察其干扰效果，为探索肿瘤基因治疗的新途径奠定基础。方法设计针对Stat3编码区的有短发夹结构的两条DNA序列，经退火成互补双链，克隆到载体Pavu6＋27中构建重组体Pavu6＋27-Stat3，再对重组质粒进行酶切鉴定、DNA测序分析。脂质体法转染重组体至大肠癌HCT116细胞株中，以空质粒（Pavu6＋27）转染为对照；RT-PCR法观察Stat3基因表达改变。结果酶切鉴定和测序分析均提示Stat3靶向RNA干扰重组体的构建成功，Pavu6＋27-Stat3转染后HCT116细胞Stat3基因表达较空载体Pavu6＋27转染的对照组显著下降。结论Stat3靶向RNA干扰重组体成功构建，并能有效抑制HCT116细胞中Stat3基因表达。本实验为下一步利用RNAi技术沉默肿瘤细胞中Stat3基因的表达，诱导肿瘤细胞凋亡或抑制其增生，为探索肿瘤基因治疗的新途径打下基础。     

新疆阿魏树脂不同分离部位对结肠癌细胞HCT116的抑制作用

目的: 分析新疆阿魏树脂不同分离部位对结肠癌细胞HCT116抑制作用的活性,并确定其有效活性部位。 方法: 通过磺酰罗丹明B比色法（SRB）与流式细胞术以细胞密度1×10⁵个mL,药物质量浓度250,125,62.5,31.25,15.6 mg·L^-1（流式细胞术药物浓度为62.5 mg·L^-1）,检测新疆阿魏树脂不同分离部位（石油醚部位、乙酸乙酯部位,甲醇部位）对结肠癌细胞 HCT116药物作用24 h后的增殖抑制与凋亡作用,以IC₅₀和总凋亡率作为指标衡量其各分离部位抑制肿瘤的活性效能。 结果: 石油醚部位:对结肠癌细胞HCT116增殖抑制IC₅₀ 68.7 mg·L^-1,总凋亡率为（43.4±1.1）%;乙酸乙酯部位:IC₅₀43.7 mg·L^-1, 总凋亡率为（56.2±0.9）%;甲醇部位:IC₅₀59.6 mg·L^-1,总凋亡率为（46.7±3.1）%。 结论: 新疆阿魏树脂乙醇提取物的乙酸乙酯萃取部位对结肠癌细胞HCT116细胞毒性作用较强并能促进其大量凋亡,初步确定为新疆阿魏树脂抗肿瘤活性部位。     

PED/PEA-15及其磷酸化状态在卵巢上皮肿瘤中的表达及意义

目的:探讨PED/PEA-15(phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes)蛋白及其磷酸化状态在卵巢上皮性肿瘤中的表达及临床意义。方法:免疫组化法检测40例卵巢上皮良性肿瘤(良性组)、40例卵巢交界性肿瘤(交界性组)、50例卵巢上皮癌(恶性组)中PED/PEA-15及其磷酸化状态的阳性表达水平。结果:PEA-15蛋白在良性组中表达高于交界性组及恶性组(P0.05),且在卵巢癌组中随着卵巢癌恶性程度的增高及临床分期的发展而降低(P0.05),但与患者年龄无明显相关性(P0.05)。卵巢癌中PEA-15磷酸化状态p PEA-15-Ser104蛋白表达高于交界性及良性上皮肿瘤(P0.05)。卵巢癌组织中p PEA-15-Ser104蛋白表达与组织学分级和临床分期有关(P0.05),但与有无淋巴结转移及年龄无明显相关性(P0.05);另一位点磷酸化状态p PEA-15-Ser116在不同性质卵巢组织、不同年龄、组织学分级、临床分期、淋巴结转移分组中表达无差异(P0.05)。结论:PEA-15及其磷酸化状态在卵巢癌的发生发展中发挥重要作用,可否通过调控PED/PEA-15的磷酸化状态从而使之成为卵巢癌治疗的重要靶点仍需进一步研究。     

A winged-helix protein from Sulfolobus turreted icosahedral virus points toward stabilizing disulfide bonds in the intracellular proteins of a hyperthermophilic virus

Sulfolobus turreted icosahedral virus (STIV) was the first non-tailed icosahedral virus to be isolated from an archaeal host. Like other archaeal viruses, its 37 open reading frames generally lack sequence similarity to genes with known function. The roles of the gene products in this and other archaeal viruses are thus largely unknown. However, a protein's three-dimensional structure may provide functional and evolutionary insight in cases of minimal sequence similarity. In this vein, the structure of STIV F93 reveals a homodimer with strong similarity to the winged-helix family of DNA-binding proteins. Importantly, an interchain disulfide bond is found at the dimer interface, prompting analysis of the cysteine distribution in the putative intracellular proteins of the viral proteome. The analysis suggests that intracellular disulfide bonds are common in cellular STIV proteins, where they enhance the thermostability of the viral proteome.     

Sequence Analysis Demonstrates that VP6, NSP1 and NSP4 Genes of Indian Neonatal Rotavirus Strain 116E are of Human Origin

We have sequenced the genes encoding the inner capsid protein VP6 and the nonstructural proteins NSP1 and NSP4 of the Indian neonatal serotype P8[11]G9 human/bovine reassortant candidate vaccine rotavirus strain 116E. These three genes share a high degree of sequence and deduced amino acid homology with human prototype strain Wa. Our results confirm and extend those of previous RNA-RNA hybridization studies which suggested that these genes are of human origin, and will facilitate examination of the host immune response to 116E induced by natural infection and vaccination. This revised version was published online in July 2006 with corrections to the Cover Date.     

高活性的人白细胞介素-3突变体的构建与表达

目的:在大肠杆菌中构建表达高活性的人白细胞介素-3(hIL-3)突变体。方法:通过定点突变的方法构建hIL-3突变体K116V和K116W的表达载体,并实现hIL-3突变体在大肠杆菌中的表达。对所得包涵体蛋白依次溶解、纯化、透析复性。用Westernblot检测hIL-3突变体的特异性。单溶液细胞增殖(MTS)测定hIL-3突变体的生物学活性。结果:hIL-3在大肠杆菌中高效表达,表达量占菌体总蛋白的40%以上,主要以包涵体的形式存在。将包涵体溶解在8mol/L脲中,经Ni-NTA-Sepharose柱纯化后纯度达90%以上。MTS法测定hIL-3突变体K116V的活性为野生型的5倍,K116W的生物学活性也有所增加。结论:获得了高活性的hIL-3突变体K116V,有望成为hIL-3的替代品,也证明了hIL-3第116位的赖氨酸残基对于hIL-3的生物学活性很重要。