Ectopic expression of S-RNase of <Emphasis Type="Italic">Petunia inflata</Emphasis> in pollen results in its sequestration and non-cytotoxic function

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S₂-RNase, S₃-RNase and non-glycosylated S₃-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.     

S100和GFAP在猫上丘表浅层表达差异的免疫组织化学观察

目的比较青、老年猫上丘表浅层（superricial Superior Colliculus,sSC）星形胶质细胞中S100蛋白与胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）表达的老年性变化,并探讨其在动物视觉功能衰退中的意义。方法采用免疫组织化学方法（SABC法）示青、老年猫上丘表浅层S100免疫阳性反应（S100-immunoreactive,S100-IR）细胞及胶质纤维酸性蛋白免疫反应阳性（GFAP-immunoreactive,GFAP-IR）细胞。光镜下观察、拍照,并利用Image-ProExpress图像分析软件对上丘表浅层各层S100和GFAP免疫反应阳性细胞密度及其灰度值进行测量。结果与青年猫相比,老年猫上丘表浅层中S100蛋白与GFAP表达均有不同程度的显著增强（P〈0.01）。结论衰老进程中,上丘表浅层出现S100、GFAP表达增强,星形胶质细胞存在明显的反应性活化与增生,这对维持上丘表浅层神经元的活性和神经元之间的通讯联系,从而延缓老年性视觉功能衰退具有重要意义。     

1．3倍体HBV YIDD变异株真核表达载体构建及鉴定

构建HBV YIDD拉米夫定耐药株1．3倍全基因真核表达载体,为进一步探讨乙肝病毒变异株的生物学特性及筛选抗病毒药物奠定基础。参考GenBankHBV序列设计并合成一系列引物,以临床证实为拉米夫定耐药的病人HBV DNA为模板,通过PCR扩增得到HBV全基因组并克隆至pGEM—T Easy载体中,经测序证实聚合酶基因存在YIDD变异,然后以该病人的HBV全基因组为模板构建1．3倍全基因HBV—YIDD变异真核表达载体pcDNA3．1（＋）-1．3HBV。通过PCR扩增,酶切及测序证明pcDNA3．1（＋）-1．3HBV表达载体构建成功,该表达载体的构建为后期建立稳定表达HBV—YIDD变异的细胞模型提供材料。     

氨基酸强化饵料对大鲮鲆诱食活性的研究

以平均体长为3~5 cm大鲮鲆为实验对象,采用触球法、迷宫法等实验方法,通过8种氨基酸对实验大鲮鲆进行诱食活性试验研究,每组实验重复3次,使用SPSS软件进行分析。结果表明,其中L-赖氨酸、L-甘氨酸、L-组氨酸的诱食活性呈显著性差异(P<0.01)。其中以甘氨酸的诱食活性最大,其它次之。     

鼻咽癌组织中氧化性DNA损伤标志物8-硝基鸟嘌呤和8-羟基脱氧鸟苷的检测及与iNOS的关系

8-硝基鸟嘌呤（8-nitroguanine, 8-NitroG）和8-羟基脱氧鸟苷（8-hydroxy-2′-deoxyguanosine, 8-OHdG）是2个氧化性DNA损伤生物标志物,而诱导型一氧化氮合酶(iNOS)在病理状态下催化细胞合成与氧化性DNA损伤有关的 氧自由基NO.本研究通过检测鼻咽癌组织中8-NitroG、8-OHdG和iNOS的免疫反应强度,初步探究鼻咽癌的发生和发展是否与氧化性DNA损伤有关以及8-NitroG、8-OHdG与iNOS表达的关系.利用多克隆抗体8-NitroG和单克隆抗体8-OHdG、iNOS,采用双色荧光免疫组织化学方法检测鼻咽癌组织中8-NitroG、8-OHdG和iNOS的免疫反应,秩和检验统计学方法分析鼻咽癌和慢性咽炎鼻咽组织之间8-NitroG、8-OHdG和iNOS免疫反应强度的差异.结果显示,19例鼻咽癌组织细胞中,8-NitroG、8-OHdG和iNOS均为强免疫反应,8-NitroG和8-OHdG阳性率100%,iNOS阳性率94.7 %,与13例慢性咽炎组织比较差异显著(P.<0.05).结果提示,鼻咽癌的发生和发展与氧化性DNA损伤有关,其原因与炎症等病理刺激下鼻咽组织高表达的iNOS催化细胞合成氧自由基NO引起的8-NitroG和8-OHdG DNA损伤密切相关.另外,8-NitroG和8-OHdG有望成为辅助鼻咽癌诊断的生物标志物.     

Cyclin D1 Induction by Benzo[a]pyrene-7,8-diol-9,10-epoxide via the Phosphatidylinositol 3-Kinase/Akt/MAPK- and p70s6k-dependent Pathway Promotes Cell Transformation and Tumorigenesis

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of B[a]P, has been well recognized as one ubiquitous carcinogen, but the molecular mechanism involved in its carcinogenic effect remains obscure. In the present study, we found that bronchial epithelial cells (Beas-2B) and hepatocytes treated with B[a]PDE presented a significant increase of cyclin D1 expression. Moreover, Akt, p70^s6k, and MAPKs including JNK, Erks, and p38 were notably activated in B[a]PDE-treated Beas-2B cells, whereas NF-κB, NFAT, and Egr-1 were not. Our results demonstrated that JNK and Erks were required in B[a]PDE-induced cyclin D1 expression because the inhibition of JNK or Erks by a selective chemical inhibitor or dominant negative mutant robustly impaired the cyclin D1 induction by B[a]PDE. Furthermore, we found that overexpression of the dominant negative mutant of p85 (regulatory subunit of phosphatidylinositol 3-kinase) or Akt dramatically suppressed B[a]PDE-induced JNK and Erk activation as well as cyclin D1 expression, suggesting that cyclin D1 induction by B[a]PDE is via the phosphatidylinositol 3-kinase/Akt/MAPK-dependent pathway. In addition, we clarified that p70^s6k is also involved in B[a]PDE-induced cyclin D1 expression because rampamycin pretreatment dramatically reduced cyclin D1 induction by B[a]PDE. More importantly, we demonstrated that up-regulated cyclin D1 by B[a]PDE plays a critical role in oncogenic transformation and tumorigenesis of Beas-2B cells. These results not only broaden our knowledge of the molecular mechanism of B[a]PDE carcinogenicity but also lead to the further study of chemoprevention of B[a]PDE-associated human cancers.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

Modifications of nonwoven polyethylene terephthalate fibrous matrices via NaOH hydrolysis: Effects on pore size, fiber diameter, cell seeding and proliferation

A simple NaOH treatment method was developed for fabricating nonwoven fibrous matrices of polyethylene terephthalate (PET) with predictable porosity, pore size, and fiber diameter. Matrices with various porosities (90–97%), fiber diameters (13.5–25 μm), and pore sizes (54–65 μm) were prepared by treating with 1N NaOH at 70 °C for up to 120 h, resulting in up to 70% hydrolysis of the PET polymer. The hydrolysis of PET polymer by NaOH was found to follow a second-order kinetics with respect to the fiber surface area. Accordingly, mathematical models were developed to predict matrix porosity, fiber diameter, and apparent pore size of the PET matrices. The exponential decay coefficient of PET polymer was found to be 0.0147 h⁻¹. The matrices were used to study the effects of pore size and fiber diameter on cell seeding and proliferation. The seeding study demonstrated that cell adhesion on PET fibers can be enhanced, largely due to the increased surface roughness of the PET fibers. Decreasing the fiber diameter increases the surface curvature of the fibers and decreases available surface area for cell attachment, which, however, only resulted in a small decrease in the cell growth rate.