Detection of circulating tumor cells in the cerebrospinal fluid: A new frontier

Comment on: Patel AS, et al. Oncotarget 2011; 2:752-760     

Compound Heterozygosity of the Functionally Null Cdh23v-ngt and Hypomorphic Cdh23ahl Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23^ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23^v-ngt) null allele with mice carrying the hypomorphic Cdh23^ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23^v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23^ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23^v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23^v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K_i and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

华癸根瘤菌外膜孔蛋白编码基因MCHK_1326突变株的构建与共生固氮表型分析

【目的】Mesorhizobium huakuii 7653R的MCHK_1326基因编码一种外膜孔蛋白,可能参与根瘤菌侵染宿主植物以及结瘤固氮过程,本研究旨在探索该基因在共生固氮中的功能。【方法】生物信息学分析MCHK_1326蛋白的结构特征及生物学功能,启动子原位表达技术检测MCHK_1326共生时空表达特征,利用Cre-loxp系统构建MCHK_1326缺失突变株,考察其共生固氮表型及早期侵染事件,通过植物盆栽并额外添加无机氮源,检测突变株接种紫云英后的共生固氮表型变化。【结果】MCHK_1326基因在侵染早期如侵染线的延伸等过程中表达,在成熟根瘤的侵染区表达,与野生型相比,突变体△1326侵染线和根瘤原基数量显著减少;植株地上部分鲜重与固氮酶活性极显著降低,根瘤数量和根瘤重量显著降低;额外添加无机氮源能恢复其共生缺陷表型。【结论】MCHK_1326基因参与根瘤菌早期侵染和结瘤,在根瘤发育与共生固氮过程中发挥作用。     

核桃细菌性黑斑病菌rpfG基因的功能分析

[目的] 本研究旨在揭示核桃细菌性黑斑病菌（Xanthomonas arboricola pv.juglandis,Xaj） DW3F3中rpfG基因的生物学功能,从而为核桃细菌性黑斑病防治药剂的开发提供作用靶点。[方法] 以野油菜黄单胞菌（Xanthomonas campestris pv.campestris,Xcc）8004菌株以及水稻白叶枯病菌（Xanthomonas oryzae pv.oryzae,Xoo） PXO99^A的rpfG基因为模板序列,对Xaj野生型菌株DW3F3的基因组序列进行检索。利用同源重组技术,对Xaj中rpfG基因进行敲除,并用生物化学方法对基因缺失菌株的相关毒力因子、抗逆性进行检测。[结果] 通过同源比对,在XajDW3F3的基因组中发现了与XccrpfG、XoorpfG同源的基因,并成功获得rpfG的缺失突变株ΔrpfG。与野生型相比,突变株ΔrpfG的生物被膜形成能力仅为野生型XajDW3F3的44.58%;胞外多糖产量也由野生型的8.47 mg/mL降为5.23 mg/mL;ΔrpfG的絮凝活性增加,能使菌液变澄清;运动性实验显示ΔrpfG的运动直径比野生型增加了12.38%;胞外酶的分泌也发生了不同程度的改变,突变株分泌纤维素酶的能力极显著降低,淀粉酶活性有所提高,而分泌蛋白酶的能力未发生变化;此外rpfG缺失后,Xaj对逆境（盐、酸、SDS、硫酸铜）的耐受力降低。[结论] 结果表明rpfG基因能影响核桃细菌性黑斑病菌的致病相关性状,并赋予了细菌一定的抗逆性。     

截短型rBTI的表达、纯化及其对EC9706细胞生长的抑制作用

依据先前获得的重组荞麦胰蛋白酶抑制剂（rBTI）氨基酸序列及三维分子构像,分别构建了rBTI C末端缺失VVM、TPVVM或VDTPVVM的截短型pExsecI BTI t1,pExsecI BTI t2和pExsecI BTI t3重组质粒.转入大肠杆菌BL21中进行表达,并通过Resource Q阴离子交换层析分离.实验结果显示,3个工程菌均以可溶方式表达,目的蛋白在SDS PAGE图谱中显示单一条带,其纯度达98％以上. 理化性质分析表明,截短型rBTI与野生型rBTI具有相似的胰蛋白酶抑制活性,并具有很好的热稳定性及酸碱稳定性. 将野生型和截短型rBTI分别作用于人食管癌EC9706细胞.MTT检测发现,C末端截短不同数目的氨基酸后,与野生型rBTI相比,在相同浓度下截短型rBTI仍具有一定的抑制肿瘤细胞生长作用,其抑制作用范围是截短前的50%左右. 这些结果提示, rBTI的 C末端氨基酸残基缺失未引起活性区域或功能部位的较大改变,从而保留了其对胰蛋白酶的抑制作用和部分生物学功能.     

粘液繁殖体种子的粘液质形成、分泌及释放相关基因

种皮粘液质是在种皮外层细胞的高尔基体内产生并分泌到胞腔内或细胞壁层的一种果胶类多糖物质.当干燥种子遇水后,粘液质即刻被释放形成透明胶质并完全包被整个种子.粘液质对种子的扩散定居、种子萌发以及幼苗的存活和生长均具有重要作用.粘液质作为一种模型研究细胞壁的产生及其形成的分子机制已经成为植物种皮发育与环境变化相适应关系的研究...     

重组蓖麻、相思子毒素A链嵌合突变体的制备与分析

目的:构建蓖麻毒素(RIC)、相思子毒素(ABR)A链突变体的嵌合体蛋白,实现嵌合体蛋白的可溶性表达、纯化及抗原性分析。方法:采用柔性linker连接RIC A链突变体(mRICAD75AV76MY80A)和ABR A链突变体(mABRAE164AR167L),构建嵌合体基因mRICA/mABRA,将该嵌合体基因亚克隆至原核载体pQE80L构建表达质粒pQE80L-mRICA/mABRA,再转化至大肠杆菌M15获得表达工程菌株M15/pQE80L-mRICA/mABRA,工程菌在18℃经0.1 mmol/L的IPTG诱导14 h,表达的嵌合体蛋白经Ni-NTA亲和层析柱纯化,通过ELISA和Western印迹检测嵌合体蛋白的抗原性。结果:所获得的mRICA/mABRA嵌合体基因经一致性比对分析,与预计嵌合基因的序列一致性为100%,其开放读框全长1572 bp,编码524个氨基酸残基;重组表达质粒pQE80L-mRICA/mABRA经PCR及双酶切鉴定证明构建正确,嵌合体蛋白相对分子质量约为62×103,与预测相符,可溶性的嵌合体蛋白经Ni-NTA亲和层析柱纯化,纯度可达99%;间接ELISA和Western印迹结果表明,嵌合体蛋白能同时与抗RIC多克隆抗体和抗ABR多克隆抗体发生特异的抗原抗体反应。结论:得到的mRICA/mABRA嵌合体蛋白具有良好的抗原性,为研制新型RIC和ABR双价疫苗奠定了重要基础。     

Selection and characterization of a 7‐mer peptide binding to divalent cations

A 7‐mer peptide (S‐T‐L‐P‐L‐P‐P) that bound to various divalent cations was selected from a phage display peptide library. Isothermal calorimetric analysis revealed that the peptide bound to Pb²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. Through the use of CD studies, no secondary structural changes were observed for the peptide upon binding to divalent cations. Ala scanning mutant peptides bound to Hg²⁺ with a reduced affinity. However, no single substitution was shown to affect the overall affinity. We suggest that Pro residues chelate divalent cations, while the structure formed by the peptide is also important for the binding process. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.