肺癌靶向多肽荧光分子探针的研制及其应用

目的:选择能与整合素α3受体结合的小分子多肽cNGQGEQc-L作为靶向载体，将羧基荧光素（FAM）与cNGQGEQc-L连接构建荧光分子探针，通过荧光成像探讨荧光多肽分子探针用于肺腺癌显像的可行性。方法:利用倒置荧光显微镜观察FAM-cNGQGEQc-L与肺腺癌A549细胞结合部位，流式细胞仪检测荧光多肽与A549细胞的竞争抑制实验，观察FAM-cNGQGEQc-L随浓度的增加与肺腺癌A549细胞结合能力的变化情况。通过小动物活体成像仪，观察荧光多肽在荷瘤裸鼠体内的生物分布特点。结果:倒置荧光显微镜显示荧光多肽cNGQGEQc-L能与A549细胞结合，结合部位在细胞膜和细胞质中。流式细胞仪测试结果证明荧光多肽与A549细胞的结合具有特异性和饱和性，当FAM-cNGQGEQc-L浓度为0.125 mmol/L时，荧光多肽与A549细胞的结合趋近饱和。荷瘤裸鼠活体成像显示移植瘤能够摄取荧光多肽，且荧光多肽通过泌尿系统和胆道系统排泄。结论:体外、体内荧光实验结果显示，荧光多肽分子探针FAM-cNGQGEQc-L可与肺腺癌A549细胞、肺癌移植瘤结合，能够特异性靶向肺腺癌。     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Subtyping of MRSA isolates belonging to a widely disseminated clonal group by polymorphism of the dru sequences in mec-associated DNA

During the past 7 years the "Berlin epidemic MRSA" has spread throughout whole Germany. SmaI macrorestriction patterns of this clonal group are rather stable as are the length polymorphisms at the 3' end of the coagulase gene and the x-region of spa. However, the dru region (direct repeat units) of mec-associated DNA exhibits a length polymorphism due to deletion of one or more direct repeat units. Five different subclones could be discriminated by dru region length polymorphism. Location of deletions and of a few point mutations allow a delineation of these subclones.     

Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin B-positive (A- B+) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A- B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A/B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A- B+ strains had a c. 1.7-kb deletion in the 3'-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A- B+ isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A- B+ isolates belonged to PCR-ribotype 017. A- B+ C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.     

空肠弯曲菌HSP43诱导自身免疫机理──分子模拟和抗原优势

我们曾发现空肠弯曲菌４３ｋＤ热休克蛋白（ＨＳＰ４３）能诱导小鼠产生自身免疫应答，本文则进一步分析了这种诱导作用的机理。二株针对ＨＳＰ６０保守区序列的单克隆抗体ＩＩＨ９和ＭＬ－３０不但能结合人及小鼠细胞中ＨＳＰ６０家族成员，而且也能结合ＨＳＰ４３，表明ＨＳＰ４３、人及小鼠ＨＳＰ６０三者均属同一家族成员，具有序列上的高度同源性。小鼠用空肠弯曲菌免疫后出现了针对１０种菌体蛋白的抗体，其中最早被诱导的抗体是针对ＨＳＰ４３的，并且该种抗体在随后８０ｄ观察期间内保持了较高活性，表明ＨＳＰ４３是优势抗原。本文结果提示，ＨＳＰ４３较易被免疫系统识别而产生应答，通过和宿主ＨＳＰ６０高度序列同源性而可能呈现分子模拟，从而诱导自身免疫损伤。     

The Evolving Concept of Renal Neoplasia: Impact of Emerging Molecular and Electron Microscopic Studies

The classification of renal tumors has evolved from one that initially encompassed only 2 types of tumors, i.e., clear and granular cell carcinomas, to the markedly expanded recent classification that incorporates new entities, some of which are primarily defined by specific molecular abnormalities. Despite these advances, a single tumor category, clear cell carcinoma, still incorporates the majority (∼70%) of renal tumors. It is, however, postulated that this single category is likely to encompass several different tumor types that are, at present, undifferentiated. Electron microscopic studies have been pivotal in defining the spectrum of oncocytoma-chromophobe renal cell carcinoma. Cytoplasmic eosinophilia found in some renal cell carcinomas currently classified as clear cell type is under intense study. Tumors that have recently emerged from this group include tumors with translocations involving chromosome Xp11.2, carcinomas associated with neuroblastoma and epithelioid angiomyolipoma. The spectrum of renal tumors seen in younger patients is wider than among older patients, with rare and unusual tumors being more likely seen in younger patients. The author concludes that although the routine application of electron microscopy to kidney tumor diagnosis may not be practical, systematic ultrastructural studies of these tumors may aid in the definition of new entities.     

人血清载脂蛋白H的纯化与鉴定

为了研究载脂蛋白H（apoH)与疾病的关系，分别用HClO4,(NH4)2SO4沉淀、DEAE－纤维素离子交换层析，从人血清中纯化了载脂蛋白Ho用测定分子量，分析氨基酸组成和N－端氨基酸测序三个方面进行鉴定。用SDS－PAGE测得其相对分子量对54000。用高效液相色谱测定其中15种氨基酸的相对含量（mol/10^3mol残基）如下：Asp119.8,Glu108,Thr94.8,Ser78.6,Phe64.9,Gly1008,Ala57.2,Val57.2,Met10.6,Ile50.3,Leu77.1,Tyr31.9,His15.2,L-ys81.4,Arg39.4;d PE-ABI公司生产的475A型气相蛋白质测序仪上测得其N端10个氨基酸残基顺序如下：NH2－Gly-Arg-Cys-Pro-Asp-Asp-Leu。纯化得到的apoH与肝素有高的亲和性，还能与乙肝表面抗原结合。纯化apoH的方法相对比较简单，能达到测序的要求。     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

A PCR–restriction fragment length polymorphism assay to genotype human metapneumovirus

Human metapneumovirus (hMPV) genotypes A and B show epidemiological and probably clinical differences. This report describes a fast and simple PCR–restriction fragment length polymorphism (PCR-RFLP) assay, involving digestion of the fusion protein gene with Tsp 509I, that allows lineages A1, A2, B1 and B2 to be distinguished. The assay should help in elucidating the epidemiology of hMPV, and possibly in predicting the severity of clinical infection.