Subcellullar localization of tumor-associated antigen 3H11Ag

3H11Ag, a tumor-associated antigen defined by the monoclonal antibody 3H11 that specifically recognizes cancer cells in various tumor tissues, was successfully cloned recently, but its function is unknown. To explore the potential roles it plays in tumors, we analyzed its subcellular localization in the present study. By expressing 3H11Ag fused with fluorescent protein in COS-7 cells, we found that 3H11Ag localizes to both cytoplasm and nucleus, which was confirmed by subcellular fractionation. And sequentially extracting the nuclei of COS-7 cells transfected with 3H11Ag showed that it is a DNA- and nuclear matrix-associated protein. Moreover, by expressing a series of red fluorescent protein-tagged truncated forms of 3H11Ag, it was demonstrated that the 150 amino acid residues at its C-terminal are fully responsible for the subcellular localization. In addition, the results of the computational analysis of 3H11Ag were in accordance with those of the experimental analysis. All these data would be helpful to elucidate the functions of 3H11Ag.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.     

T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS

The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.     

Fine needle aspiration cytology of adult perineal rhabdomyosarcoma: a case report

BACKGROUND: Adult perineal soft tissue sarcomas are rare. Fewer than 30 cases have been reported, and all were diagnosed after surgical resection by histologic examination. Below we report a case in which the diagnosis was established preoperatively by fine needle aspiration (FNA). CASE: A 27-year-old man presented with a firm, midline, perineal mass. Magnetic resonance imaging showed a 3-cm, enhancing mass that was considered neoplastic. FNA biopsy, followed by cytologic examination, revealed moderately cellular aspirates composed of discohesive, small, blue cells with scant cytoplasm, high nuclear/cytoplasmic ratios and pleomorphic nuclei with irregular nuclear contours; uniform, hyperchromatic chromatin; and occasional mitotic figures. Frequent naked nuclei and scattered cells with more abundant, dense cytoplasm and eccentric nuclei were also noted. The diagnosis of rhabdomyosarcoma was favored on FNA and was corroborated by immunohistochemical stains for desmin, myogenin and CD56. Upon surgical resection, the diagnosis of alveolar rhabdomyosarcoma was confirmed histologically and immunophenotypically. CONCLUSION: FNA is a useful tool in diagnosing soft tissue lessions of the perineum, including rare primary tumors, such as adult rhabdomyosarcoma. In this case, early identification avoided incisional biopsy and directed appropriate extirpative surgery and reconstruction considerations.     

大黄葸醌提取物对建鲤抗应激及生长的影响

将750尾建鲤随机分成5组,第1组为对照组,投喂基础日粮,另外4组为试验组,投喂基础日粮中分别添加0．5％、1．0％、2．0％、4．0％大黄蒽醌提取物。从2005年7月到9月连续投喂70d后,对鱼体进行高密度应激,测定其生长、应激前后血液皮质醇、血糖、溶菌酶等变化。结果表明：与对照组相比,应激前添加大黄葸醌提取物提高了鱼体增重率、特定生长率、鱼体丰满度、血液溶菌酶活性,降低了饵料系数与鱼体死亡率,但是与大黄葸醌提取物的添加水平不成线性关系,其中2．0％试验组还显著降低了血液皮质醇。高密度应激1d后,各组血液中的皮质醇、血糖、溶菌酶都有不同程度的增加,其中对照组最高,添加1．0％和2．0％大黄蒽醌提取物的试验组相对较低。应激前后各组鱼的攻毒试验表明：除应激前添加1．0％和2．0％大黄蒽醌提取物的组没有死鱼外,其它各组都有死鱼,其中对照组死亡率最高。因此添加1．0％-2．0％大黄蒽醌提取物提高了机体抗应激能力,并对病原菌感染起一定的保护作用,促进了鱼体生长。     

Association of polymorphisms in the Angiotensin-converting enzyme gene with Alzheimer disease in an Israeli Arab community

Several lines of evidence support for a role of angiotensin converting enzyme (ACE) in Alzheimer disease (AD). Most genetic studies have focused on an Alu insertion/deletion (I/D) polymorphism in the ACE gene (DCP1) and have yielded conflicting results. We evaluated the association between 15 single-nucleotide polymorphisms (SNPs) in DCP1, including the I/D variant, and AD in a sample of 92 patients with AD and 166 nondemented controls from an inbred Israeli Arab community. Although there was no evidence for association between AD and I/D, we observed significant association with SNPs rs4343 (P = .00001) and rs4351 (P = .01). Haplotype analysis revealed remarkably significant evidence of association with the SNP combination rs4343 and rs4351 (global P = 7.5 x 10(-7)). Individuals possessing the haplotype "GA" (frequency 0.21 in cases and 0.01 in controls) derived from these SNPs had a 45-fold increased risk of developing AD (95% CI 6.0-343.2) compared with those possessing any of the other three haplotypes. Longer range haplotypes including I/D were even more significant (lowest global P = 1.1 x 10(-12)), but the only consistently associated alleles were in rs4343 and rs4351. These results suggest that a variant in close proximity to rs4343 and rs4351 modulates susceptibility to AD in this community.     

过表达PKCε和PKCη在HCC1806细胞中的抗凋亡作用

为探讨PKCε、PKCη对乳腺癌细胞中TNFα诱导凋亡的影响,采用PCR技术,从人肌肉cDNA文库中克隆了全长PKCε和PKCη序列,亚克隆至pcDNA3,转化HCC1806细胞,并选择出稳定的转化细胞株.然后用流式细胞仪检测了PKCε、PKCη过表达对DNA断裂的影响,用Western印迹方法检测了PKCε、PKCη过表达对PARP、caspase 3和caspase 8水解的影响,以及对Bcl-2,Bax表达的影响,和对MAPK磷酸化的影响.流式细胞仪分析DNA含量的结果表明：TNFα处理后,HCC1806/PC、HCC1806/ε、HCC1806/η的sub_G1状态的DNA含量分别为：57.7%、23.3%、14.6%.Western印迹结果表明,当用0.01nmol/L TNFα处理,HCC1806/η中PARP、caspase3、caspase8的水解程度最低,HCC1806/ε中次之,HCC1806/PC最严重;与HCC1806/PC比较,无论用不用TNFα处理,HCC1806/ε都表达更高的Bcl_2,而HCC1806/η不影响Bcl_2的表达.过表达PKCη而不是PKCε抑制了由TNFα引起的p38和JNK的磷酸化,并且是JNK的抑制剂SP600125而不是p38的抑制剂SB220025抑制了PARP的裂解.以上结果显示,过表达PKCε、PKCη可能通过不同的信号途径在HCC1806中起抗凋亡作用,PKCε上调了bcl_2的表达,而PKCη抑制了JNK的磷酸化.     

锅柄式PCR及其在混合谱系白血病基因断裂区中的应用

染色体11q23的混合谱系白血病(MLL)基因的断裂点簇集群区(BCR)的转位,可引起婴儿急性白血病及与DNA拓扑异构酶Ⅱ抑制剂的生化治疗法相关的白血病。由于MLL基因包括大约30个不同的转位伴侣基因,几个断裂点所在的伴侣基因的序列还不清楚,因而对MLL基因断裂点的PCR克隆是很困难的。锅柄式PCR,即用已知5'端序列和未知的3'端伴侣基因序列以形似一个锅和一个柄的模板扩增断裂点基因DNA,是一种克隆MLL基因断裂区的新方法,可以扩增未知3'端序列的断裂点簇集群区。