嵌合内含子对抗bFGF抗体基因在293T细胞中表达的影响

目的：构建含嵌合内含子和抗bFGF抗体基因的真核表达载体并在293T细胞中表达,探讨嵌合内含子对抗体表达的影响。方法：设计引物分别从载体pCl-neo和pComb3-Fab25中扩增出内含子和抗体Fd段、κ链基因序列,按不同组合构建到含人免疫球蛋白IgG1恒定区Fc段基因的表达载体pIgG中。重组质粒经脂质体PEI转染293T细胞,荧光显微镜观察质粒转染情况,采用夹心ELISA和Western blot检测细胞上清中抗体的表达。结果：DNA测序和酶切分析表明,内含子和抗体基因成功插入pIgG,获得了重组质粒pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron和pIgG-Fd-intron-κ-intron。倒置荧光显微镜下可观察EGFP大量表达,Western blot分析上清中有抗体的表达,夹心ELISA检测pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron和pIgG-Fd-intron-κ-intron抗体表达量分别为1.21mg/L、0.468mg/L、7.39mg/L、0.601mg/L。结论：成功构建了含嵌合内含子和抗体基因的真核表达载体并在293T细胞中得到表达。嵌合内含子插入κ链基因5’端可提高抗体的表达,插入Fd段5’端则抑制抗体的表达。     

人外周血单个核细胞与脐带间充质干细胞共培养的AFM研究

采用原子力显微镜与倒置显微镜在细胞层次上观察了人外周单个核细胞(PBMCs)与同种异源脐带间充质干细胞(hUC-MSCs)共培养的过程,并在单细胞水平上分析了共培养前后人外周单个核细胞的形貌和生物物理性质。结果发现:共培养后贴壁人外周单个核细胞的形态发生了很大的改变,并且表面分布着大小不一的颗粒状聚合物。利用AFM高空间分辨的力位移曲线测量系统,发现共培养72h后培养上清中人外周单个核细胞、贴壁的人外周单个核细胞的粘滞力分别是单纯培养72h的人外周单个核细胞的2倍、5倍,而细胞的硬度分别是单纯培养人外周单个核细胞的1.5倍、2倍。CCK-8检测提示,共培养过程中,干细胞的生长与外周血单个核细胞的生长出现了竞争作用。通过AFM探测人外周单个核细胞与脐带间充质干细胞共培养的可视化数据,有助于更好地了解间充质干细胞与外周血单个核细胞的相互作用。     

人DFF45蛋白多克隆抗体制备及其初步应用

目的：表达、纯化带GST标签的DNA裂解因子45（DNA fragmentation factor 45 ,DFF45）融合蛋白并制备多克隆抗体。方法：构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activated Sepharose? 4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果：成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1:20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论：DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。     

北京夏植黄瓜内生真菌区系研究

为了解黄瓜植株内生真菌的区系组成及其变化,从而为进一步研究黄瓜内生真菌的生态和功能奠定基础,对采自北京延庆的不同品种和不同生育期的40株黄瓜进行了内生真菌的分离培养。经形态学鉴定和18S rDNA序列分析,分离到的1,024株内生真菌属于18属,其中Exserohilum和Neocosmospora尚未见内生真菌的报道。Alternaria、Aspergillus、Chaetomium、Cladosporium和Fusarium在各生育期和各器官普遍存在。其中,Alternaria在叶中的定殖率达47.0%,远高于在其他器官中的定殖率;Fusarium在根中的定殖率达32.5%,远高于在其他器官中的定殖率。多数真菌类群表现出不同程度的器官偏好性,有些真菌类群则只出现在特定器官中。叶和根的内生真菌类群数量和总定殖率均高于茎和果实。随着黄瓜的生长,各器官内生真菌的类群数在增加,部分真菌的定殖率也呈上升趋势,但Neocosmospora和Chaetomium在各器官中的定殖率则随植株生长呈下降趋势。     

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.     

Miscanthus subgen. Rubimons subgen. nov. (Poaceae) and Miscanthus villosus sp. nov. from southwest Yunnan,China

Miscanthus subgen. Rubimons (B. S. Sun) Y. C. Liu et H. Peng, a new subgenus of Miscanthus Anderss. (Poaceae) is established and Miscanthus (Rubimons) villosus Y. C. Liu et H. Peng, a new species of this subgenus from southwest Yunnan, China, is described and illustrated. It is closely related to M. paniculatus (B. S. Sun) S. L. Chen & Renvoize, but differs mainly by its villous panicle axis, long callus hair, lanceolate lower lemma and the consistently longer awn of the upper lemma.     

Isolation and characterization of a novel glycogen synthase kinase-3 gene, <Emphasis Type="Italic">GmGSK</Emphasis>, in <Emphasis Type="Italic">Glycine max</Emphasis> L. that enhances abiotic stress tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel glycogen synthase kinase-3 gene, GmGSK, was isolated from Glycine max. It is 1,596 bp in length with one ORF of 410 amino acids. Southern blot analysis revealed that it has at least two copies in the G. max genome. GmGSK, when transiently expressed in Nicotiana tabacum leaves, was localized in both cell membrane and cytoplasm. Northern blot analysis indicated that GmGSK is expressed in all tissues, with highest expression in the root. GmGSK can be induced by various abiotic stresses. When transformed with GmGSK, Saccharomyces cerevisiae exhibited enhanced resistance to salt and drought stress.     

A model on liquid penetration into soft material with application to needle-free jet injection

A mathematical model has been presented for a high speed liquid jet penetration into soft solid by a needle-free injection system. The model consists of a cylindrical column formed by the initial jet penetration and an expansion sphere due to continuous deposition of the liquid. By solving the equations of energy conservation and volume conservation, the penetration depth and the radius of the expansion sphere can be predicted. As an example, the calculation results were presented for a typical needle-free injection system into which a silicon rubber was injected into. The calculation results were compared with the experimental results.     

Structures of the Michaelis complex (1.2 Å) and the covalent acyl intermediate (2.0 Å) of cefamandole bound in the active sites of the Mycobacterium tuberculosis β-lactamase K73A and E166A mutants

The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active β-lactamase, BlaC, that imparts TB with resistance to β-lactam chemotherapy. The structure of covalent BlaC-β-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 ?, respectively. These structures provide insight into the details of the catalytic mechanism.     

Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H₂O₂. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H₂O₂ levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H₂O₂ production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H₂O₂ must be taken into account in interpreting effects of phenolic compounds on cells in culture.