Components of human complement system. Testing and isolation of C3 and C5

Modified reagents for testing the hemolytic activity of human complement components, C3 and C5, have been obtained. These reagents were obtained by treatment of human blood serum pools with a saturated solution of KBr (reagent R3) or 2 M KSCN and denaturated yeasts (reagent R5). These reagents were found to be rich in the serum factor obtained through the use of DEAE-cellulose DE-52 and containing the active component of the complement (C4). To test the sensitivity and specificity of the above reagents, components C3 and C5 were purified. After this procedure these components emerged as hemolytically active, electrophoretically and immunophoretically homogeneous components, C3 and C5. DEAE-cellulose DE-52, DEAE-Sephacel, Hydroxylapatite and Ultra-gel AcA-34 were used consecutively as purification agents. The activity yields of components C3 and C5 with regard to the initial serum levels were 31% and 18%, respectively.     

Opposing functions of PLAG1 in pleomorphic adenoma: a microarray analysis of PLAG1 transgenic mice

Over-expression of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1) plays a crucial role in the formation of pleomorphic adenoma, which is the most common type of salivary gland tumor. To understand the molecular mechanisms governing PLAG1-mediated tumorigenesis, we used a microarray-based approach to identify PLAG1 target genes. We validated the expression of several genes, including Bax, Fas, p53, p21, p16, Cyclin D1, Egfr, Trail-R/DR5, c-Fos, c-myc and Igf2, by real-time RT-PCR or western blotting. Using luciferase reporter gene assays, we determined that the promoters of Bax, Fas, p53, TRAIL-R/DR5, and c-Fos were transactivated by PLAG1. PLAG1 not only activates genes that promote cell proliferation and tumor formation but also genes that inhibit these cellular processes. Therefore, we conclude that PLAG1 may play a dual role in tumor formation.     

大鼠骨骼肌损伤后中性粒细胞、巨噬细胞和肌成纤维细胞数量分析研究

目的 研究大鼠骨骼肌损伤后中性粒细胞、巨噬细胞和肌成纤维细胞数量的变化情况,为今后骨骼肌损伤修复的病理学机制研究打下坚实的基础.方法 建立大鼠骨骼肌机械性损伤动物模型,随机分为伤后6h、12h、1d、3d、7d、10d、14d及正常对照组.应用免疫组织荧光染色和免疫组织化学染色,检测大鼠骨骼肌损伤后不同时间点中性粒细胞、巨噬细胞和肌成纤维细胞的数量.结果 伤后6h-12h,损伤区可见中性粒细胞和巨噬细胞浸润,中性粒细胞数量达到高峰.伤后1d,损伤区巨噬细胞数量急剧增加,迅速达到高峰,而中性粒细胞数量开始下降.伤后3d,中性粒细胞和巨噬细胞数量都显著下降.伤后7d,肌成纤维细胞开始出现.到伤后10d-14d,损伤区主要以肌成纤维细胞为主,偶见巨噬细胞.结论 大鼠骨骼肌损伤区中性粒细胞、巨噬细胞和肌成纤维细胞数量呈时间规律性变化,以期为骨骼肌损伤修复的病理学机制研究提供参考资料.     

A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

Isolation of a cadmium-releasing bacterium and characterization of its novel protease

Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50 degrees C and pH 7.0, and was enhanced in the presence of Ca(2+), Mg(2+) and Mn(2+), while being strongly inhibited by Co(2+). The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.     

Crystal structure of quinone‐dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates

The quinone‐dependent alcohol dehydrogenase (PQQ‐ADH, E.C. 1.1.5.2) from the Gram‐negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three‐dimensional (3D) structures of the native form, with PQQ and a Ca²⁺ ion, and of the enzyme in complex with a Zn²⁺ ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ‐ADH displays an eight‐bladed β‐propeller fold, characteristic of Type I quinone‐dependent methanol dehydrogenases. However, three of the four ligands of the Ca²⁺ ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ‐ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ‐dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.     

L‐Cysteine capped CdTe–CdS core–shell quantum dots: preparation,characterization and immuno‐labeling of HeLa cells

Functionalized CdTe–CdS core–shell quantum dots (QDs) were synthesized in aqueous solution via water‐bathing combined hydrothermal method using L‐cysteine (L‐Cys) as a stabilizer. This method possesses both the advantages of water‐bathing and hydrothermal methods for preparing high‐quality QDs with markedly reduced synthesis time, and better stability than a lone hydrothermal method. The QDs were characterized by transmission electronic microscopy and powder X‐ray diffraction and X‐ray photoelectron spectroscopy. The CdTe–CdS QDs with core–shell structure showed both enhanced fluorescence and better photo stability than nude CdTe QDs. After conjugating with antibody rabbit anti‐CEACAM8 (CD67), the as‐prepared l ‐Cys capped CdTe–CdS QDs were successfully used as fluorescent probes for the direct immuno‐labeling and imaging of HeLa cells. It was indicated that this kind of QD would have application potential in bio‐labeling and cell imaging. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of NaYF(4):Yb,Er/single-walled carbon nanohorns nanocomposite and its application as cells label

A novel nanocomposite synthesis method of amino-modified NaYF(4):Yb,Er upconversion luminescent nanoparticles and single-walled carbon nanohorns was developed via covalent linkage for the first time. The nanocomposite was covalently coupled with rabbit anti-CEA8 antibody and then used successfully as a cell labeling agent for the immunolabeling and imaging of HeLa cells.     

Targeted Delivery of GDNF through the Blood–Brain Barrier by MRI-Guided Focused Ultrasound

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.     

黄芩甙对肝癌细胞BEL-7402形态学的影响

目的观察黄芩甙对肝癌细胞BEL-7402凋亡的影响,同时观察对肝癌细胞形态及超微结构、线粒体超微结构、线粒体膜电位和细胞内Ca^2＋的影响,探讨线粒体损伤在黄芩甙诱导肝癌细胞凋亡中的作用及可能的机制。方法应用细胞培养技术培养肝癌细胞BEL-7402,光镜、倒置显微镜、扫描电镜、透射电镜观察细胞形态及超微结构的变化尤其是线粒体的变化,应用流式细胞仪检测细胞凋亡百分率及线粒体膜电位、细胞内Ca^2＋的改变,免疫组化法检测细胞Bcl-2、Pax蛋白表达。结果黄芩甙诱导肝癌细胞BEL-7402凋亡呈剂量依赖关系,细胞形态、超微结构及线粒体超微结构出现明显改变,降低肝癌细胞线粒体膜电位,使细胞内Ca^2＋增加,细胞Pax表达增加,广泛分布于胞核和胞质中,Bcl-2表达减少。结论黄芩甙诱导肝癌细胞BEL-7402凋亡,线粒体损伤在黄芩甙诱导肝癌细胞凋亡中起重要作用,其机制可能为抑制肝癌细胞Bcl-2蛋白表达,促进Pax蛋白表达及细胞内Ca^2＋增加,激发线粒体膜通透性转运孔开放,线粒体跨膜电位降低,使肝癌细胞凋亡。