抗菌肽CM4抗K562癌细胞的超微结构研究

报道了家蚕抗菌肽CM4抗K562癌细胞的体外实验研究.结果表明：纯化后的家蚕抗菌肽CM4对培养的K562(人髓样白血病细胞)有很强的杀伤作用.用扫描和透射电镜观察超微结构以及用激光共聚集显微断层图像分析,表明微量纯化的抗菌肽CM4能使K562癌细胞产生一系列的病理变化,可造成细胞高度肿胀,膜与胞质分离,细胞器和膜结构排列紊乱,细胞表面微绒毛消失,出现不规则的孔洞,细胞骨架严重破坏,膜局部结构破裂,缺损,胞浆内容物大量外泄,最终细胞解体,崩解成碎片.     

柿子滩遗址第9地点出土的动物烧骨

柿子滩遗址第9地点（S9）位于山西省吉县柏山寺乡高楼河村黄河支流的清水河畔,西距黄河约7 km。从2000年发现至今,该遗址前后历经三次发掘,出土大量细石叶制品、动物化石、数件蚌制品、骨针及磨制石器等。本文重点对S9地点第4 层（12,575-11,600 cal. BP）及第5层（13,000 cal. BP）出土的动物遗存,尤其是其中测量尺寸在2cm以下的大量烧骨进行了埋藏学与动物考古学方面的观察和分析。研究结果显示,S9地点的烧骨是古人类烧烤猎物、维护遗址（甚至可能还包括以骨骼作燃料）等生存行为活动的文化残留。此外,S9地点出土烧骨的空间分布分析表明,古人类在上述行为活动之后,可能又将烧灼后的残存骨骼（与灰烬等）清理出火塘并堆放在其核心生活区的周边位置。     

The first BAFF gene cloned from the cartilaginous fish

B-cell activating factor (BAFF), a member of the TNF family, is critical to the survival, proliferation, maturation, and differentiation of B-cells. In the present study, a CpBAFF was amplified from the white-spotted catshark (Chiloscyllium plagiosum) using RT-PCR and RACE (rapid amplification of cDNA end) techniques. To our knowledge, this is the first report of any BAFF gene being cloned from a cartilaginous fish. The open reading frame (ORF) of CpBAFF cDNA consists of 819 bases encoding a protein of 272 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other identified BAFF homologues. Sequence alignment showed that CpBAFF shares 37-57% identity with BAFF amino acid sequences reported in other vertebrates. Three-dimensional structure modeling analysis revealed a soluble mature portion of CpBAFF (CpsBAFF) with a long D-E loop specific to the BAFF gene, which has not been found in other reported TNF proteins. Phylogenetic reconstruction showed that CpBAFF is most closely related to other fish BAFFs and clusters with BAFF genes from higher vertebrates (reptiles, birds, and mammals). Real-time quantitative RT-PCR demonstrated that CpBAFF mRNA expression was high in the spleen but moderate in the kidney and branchia. Recombinant CpsBAFF fused to NusA-His(6)-tag was efficiently expressed in Escherichia coli BL21 (DE3), and a molecular weight of approximately 83 kDa was determined using SDS-PAGE and Western blotting. In vitro MTT assay indicated that the purified pET43.1a (+)-CpsBAFF protein can co-stimulate the proliferation of mammalian B-cells with anti-IgM in a dose-dependent manner. The present findings not only present novel information that may be relevant to shark immunity but also provide some new insights into the origins and evolution of immunity in all vertebrates.     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.     

Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.     

水洞沟遗址第7地点动物化石初步研究

水洞沟遗址第7地点2003~2005年发掘出土动物化石2000余件。初步鉴定包括蒙古野驴(Equus hemionus)、披毛犀(Coelodonta antiquitatis)、普氏原羚(Procapra przewalskii)、水牛(Bubalus sp.)等十多个动物属种。该遗址出土的动物骨骼标本表面少见食肉类啃咬痕迹且未有啮齿类啃咬痕迹及水流磨蚀等现象,表明这一动物骨骼组合应当不是食肉类、啮齿类与水流等自然性营力带入遗址的。遗址动物群中一定比例的具切割痕骨骼标本的出现表明了古人类在动物群富集过程中的主导地位。动物考古学观察表明,处于旧石器时代晚期的该地点古人类主要对遗址附近的大中型食草类动物进行了猎捕与肢解、利用,这与相对更晚阶段的第12地点古人类以小型哺乳动物为主要猎食对象的生存策略形成了较为明显的对比。     

宁夏水洞沟遗址第7地点发掘报告

水洞沟遗址第7地点发现于2002年,位于水洞沟遗址核心区,2003年至2005年共进行三次系统发掘,揭露35个水平层,面积25m2,出土包含石制品、动物化石、鸵鸟皮及装饰品等文化遗物上万件。遗址埋藏在边沟河左岸第二级基座阶地,文化层为灰白-灰黄-灰绿色粉砂及黏土质粉砂,厚度在3m以上,遗址堆积后期局部受到小规模水流改造,但石制品组合基本保留了制作完成后的总体面貌。石制品原料取自附近的河流和湖泊成因的砾石;石制品是一个包含石核、废片、石器、砸击品和打制工具但以废片为主体的组合,个体以小型居多;锤击法为剥片的基本方法,砸击法被偶尔使用,刮削器为石器的主要类型,石器为锤击法简单修理而成。石器工业总体显示北方小型石片石器传统。光释光年代初步测定表明古人类在该遗址活动的时间大致发生在27~25ka BP,属旧石器时代晚期。