Bacterial ghosts (BGs)—Advanced antigen and drug delivery system

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2–3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.     

搞好"病原生物学"教学及促进学科发展的实践与体会

将“医学微生物学”和“人体寄生虫学”整合为“病原生物学”符合现代医学教育的取向。中国医科大学是将两个教研室从形式到实质性合并为“病原生物学”教研室，并且将两门课完全融合成一门课程和一套教师队伍进行教学的少数院校之一。本文总结了在这一新学科教学和促进学科发展中的经验和体会。     

Factor Xa-activated whole blood clotting time (Xa-ACT) for bedside monitoring of dalteparin anticoagulation during haemodialysis.

BACKGROUND: Low molecular weight heparins (LMWH) like dalteparin are increasingly used for anticoagulation during haemodialysis (HD). The available laboratory tests for monitoring LMWH anticoagulation are time-consuming and expensive, and the suitability of the conventional activated clotting time (ACT) is controversial. A simple and cheap bedside test would be useful. METHODS: We studied the factor Xa-activated whole blood clotting time (Xa-ACT) in vitro and in vivo in nine patients undergoing chronic HD with i.v. dalteparin bolus anticoagulation and compared it with the conventional ACT. Plasma anti-factor Xa (antiXa) activity was determined with a chromogenic assay. Thrombin-antithrombin complexes were measured to detect coagulation activation. RESULTS: Xa-ACT and ACT were prolonged with rising dalteparin concentration. In vitro, both clotting times were strongly correlated with the antiXa levels (r = 0.94 and 0.89, respectively). Nevertheless, compared with the ACT, the Xa-ACT was considerably more sensitive to the LMWH in vitro (healthy blood: Xa-ACT 90 s/U vs ACT 26 s/U; uraemic blood: Xa-ACT 96 s/U vs ACT 31 s/U) as well as in vivo (Xa-ACT 81 s/U vs ACT 22 s/U) and reflected different intensities of anticoagulation. An initial dalteparin bolus of 80+/-11 U/kg body weight was able to prevent coagulation activation for up to 4 h of HD. CONCLUSION: For monitoring LMWH anticoagulation the Xa-ACT was superior to the conventional ACT in vitro as well as in vivo during HD. The Xa-ACT can be useful as a LMWH bedside test. The ACT was not sensitive enough to serve as a LMWH monitoring tool.     

川产贝母种质资源遗传多样性的ISSR分析

目的:为阐明川产贝母资源间亲缘关系提供更多证据.方法:利用ISSR分子标记技术对川产10个种1变种共19份材料进行分析,采用NTSYS软件计算材料间相似系数,并用UPGMA法构建了系统树.结果:从35个ISSR引物中共筛选出11个多态性明显、反应稳定的引物,在19份供试材料DNA中共扩增出179条谱带,其中多态性条带163条,占86.8%.材料间ISSR标记遗传相似性系数(GS)在0.569～0.855.聚类分析结果显示,所有供试材料均可区分开,并聚为4组.第1组包括川贝母、甘肃贝母、长腺贝母和短丝贝母,第Ⅱ组由暗紫贝母和浓蜜贝母组成,槽鳞贝母、浙贝母、瓦布贝母和梭砂贝母聚为第Ⅲ组,湖北贝母单独为第Ⅳ组.试验结果还表明,来源于同一地区的多数川产贝母材料可分别聚在一起.结论:ISSR标记适用于川产贝母资源的遗传多样性研究,但药典收载的川贝母药材的4种基原植物种间及其与其他川产贝母属各物种间尚不能仅通过采用ISSR标记技术进行分子鉴定.此外,ISSR聚类结果同川产贝母资源的地理分布有一定关系.     

In Vivo Molecular Imaging Characterizes Pulmonary Gene Expression During Experimental Lung Transplantation

Experimental gene therapy is a promising strategy to prevent ischemia-reperfusion (I/R) injury and allograft rejection after lung transplantation, and methods will eventually be needed to characterize pulmonary transgene expression in vivo in humans. Therefore, we studied positron emission tomography (PET) as a means of performing in vivo molecular imaging in rodent models of lung transplantation. Rats were transfected endotracheally with adenovirus encoding a fusion gene of a mutant Herpes simplex virus-1 thymidine kinase and the green fluorescent protein gene (the former serving as an imaging reporter gene). Twenty-four hours after transfection, lungs were transplanted in groups representing normal transplantation, I/R injury and acute allograft rejection. Imaging was obtained either 24 h after transplantation to study reperfusion injury or 4 days after transplantation to study graft rejection. After imaging, lungs were excised and analyzed for thymidine kinase activity. Imaging detected transgene expression in transplanted lungs even in the presence of acute rejection or I/R injury. The PET imaging signal correlated with in vitro lung tissue assays of thymidine kinase activity (r(2) = 0.534). Thus, noninvasive molecular imaging with PET is a feasible, sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation.     

A binding study of phospholipase A2 with lecithin,lysolecithin and their tetrahedral intermediates using molecular modeling

We used molecular modeling to examine the binding of 1, 2-dioctanoyl-sn-glycero-3-phosphocholine (a lecithin), 1-octanoyl-sn-glycero-3-phosphocholine (a lysolecithin) and their tetrahedral intermediates in the catalytic site of phospholipase A₂ (PLA₂). We performed energy minimization on each complex, computed the binding energy, determined the relative binding energy among the complexes and calculated the difference in inter- and intramolecular energies of the components in the complexes. We found that the calculated orientation of the sn-1 ester bond of lysolecithin in the active site is similar to that of the sn-2 ester bond in lecithin, thus permitting PLA₂ to hydrolyze lysolecithin using the same mechanism as it uses to hydrolyze lecithin. On the other hand, the binding of lecithin is energetically more favorable by 4.5 kcal/mol than the binding of lysolecithin to the enzyme, and the binding of the lecithin tetrahedral intermediate is also energetically more favorable by 19.7 kcal/mol than the binding of the lysolecithin tetrahedral intermediate to the enzyme, which explains why lecithin is a better substrate than lysolecithin in the catalytic site. These results indicate that the activation energy for the hydrolysis of lysolecithin is higher than that for lecithin, consistent with the observed slower rate for the hydrolysis of lysolecithin.     

Marine biodiversity as a source of chemical diversity

Total global biodiversity is estimated at between 3 and 500 × 10⁶ species of prokaryote and eukaryote organisms spread across 70 or more phyla. The marine macrofauna alone are estimated between 0.5 and 30 × 10⁶ species and represents a broader range of taxonomic diversity than that found in the terrestrial environment, which has been the traditional source of natural products. With a typical eukaryote possessing 50,000 genes, the global marine macrofauna are the source of 2.5 × 10¹⁰ to 1.5 × 10¹² primary products and an associated extensive range of secondary products. However, only a few thousand novel compounds from marine organisms have been described. These compounds have proven unique in chemical and pharmacological terms but, as yet, no therapeutic agents have resulted. Given a broader drug discovery strategy, and facilitated by technological advances, it is predicted that the characterisation of marine chemical diversity will be accelerated. Strategies for drug discovery from the virtually untapped chemical diversity of marine organisms are discussed. © 1994 Wiley-Less, Inc.     

低抗凝肝素来源低分子肝素口服制剂的研究

以生产肝素的副产品——低抗凝肝素为原料,采用亚硝酸控制解聚法制得了低分子肝素,分子量为5300,抗凝活性为39.2u/mg,测定了理化指标。以对家兔实验性血栓形成的影响为药效学指标,确定低分子肝素口服制剂的处方组成为低分子肝素、油酸、牛胆盐。研究了所制备的低分子肝素胶囊对家兔血液流变学及血栓形成的影响,并试用于部分动脉粥样硬化症志愿者,表明该胶囊可有效地改善家免和动脉粥样硬化症志愿者血液流变性质,抑制血栓形成。     

携带人胰岛素样生长因子-1重组腺病毒的构建和鉴定

[目的] 采用 Cre-LoxP同源重组系统构建并鉴定携带人胰岛素样生长因子-1( human insulin-like growth factor-1, hIGF-1)目的基因的复制缺陷重组腺病毒,为椎间盘退变的 hIGF-1基因治疗奠定基础.[方法] PCR合成 hIGF-1基因,用 pMD18-T载体克隆 hIGF-1目的基因,酶切、测序并进行 NCBI BLAST相似性在线分析.将 pMD18-T-hIGF-1和 pDNR-1r质粒分别行 EcoRⅠ和 BamHⅠ双酶切, DNA 连接酶连接双酶切产物,转化感受态大肠杆菌 DH5α,合成中间载体 pDNR-hIGF-1,酶切鉴定.Cre-loxP系统介导 pDNR-hIGF-1和 pInt.AV1.SpaT同源重组形成 pInt.AV1.SpaT-hIGF-1重组表达质粒,行 EcoRⅠ酶切鉴定.复苏 293细胞,将 pInt.AV1.SpaT-hIGF-1和 pInt.B.B质粒在 293细胞内进行同源重组成含 hIGF-1基因的复制缺陷重组腺病毒.倒置显微镜观察 293细胞病变样效应( cytopathogenic effect, CPE);透射电镜观察 293细胞中的病毒颗粒;提取细胞裂解液中病毒 DNA, PCR鉴定 hIGF-1目的基因的存在; Western blot 检测 hIGF-1蛋白表达.用半数组织培养感染量 (tissue culture infectious dose 50,TCID50)方法测定重组腺病毒的滴度.[结果]克隆的 hIGF-1目的基因经 NCBI BLAST相似性在线分析证实与 Homo sapiens IGF-1 (GI:19923111)完全相同.pInt.AV1.SpaT-hIGF-1酶切鉴定,出现 5.4 kb和 1 kb两条片段,与理论值完全相符.转染 293细胞 12～ 14 d后,大部分细胞出现肿胀,脱落等细胞病变样效应.PCR鉴定细胞裂解液中含有 hIGF-1目的基因.Western blot 证实重组腺病毒表达 hIGF-1蛋白.第 2代腺病毒的滴度为 80× 106 PFU/mL.[结论]成功构建出含有 hIGF-1目的基因的复制缺陷重组腺病毒.