Interleukin-18 expression induced by Epstein-Barr virus-infected cells

Human Epstein-Barr virus (EBV)-negative Burkitt lymphomas cells usually grow as malignant subcutaneous tumors in athymic mice, but these tumors regress when the Burkitt cells are injected in conjunction with EBV-positive lymphoblastoid cells or when the Burkitt cells are transfected with the EBV latent membrane protein-1 (LMP-1) gene. Tumor regression is mediated, in part, by murine interferon gamma (IFN-gamma) and the IFN-gamma-induced murine chemokine IFN-gamma-inducible protein-10 (IP-10). The mechanisms by which EBV-LMP-1 promotes the expression of IFN-gamma has remained unclear. Here we show that murine interleukin (IL)-18 was consistently expressed in regressing Burkitt tumors but was either expressed at low levels or absent from progressively growing Burkitt tumors. By immunohistochemical methods, IL-18 protein was visualized in regressing but not in progressively growing Burkitt tumors. In contrast, IL-12 p35 and IL-12 p40 were only rarely expressed in regressing Burkitt tumors. In splenocyte cultures, EBV-infected lymphoblastoid cells and LMP-1-transfected Burkitt cells promoted the expression of IL-18 but not the expression of IL-12 p35 and IL-12 p40. A neutralizing antibody directed at murine IL-18 reduced murine IP-10 expression induced by EBV-immortalized cells in splenocyte cultures. These results provide evidence for IL-18 expression in response to a viral latency protein and suggest that IL-18 may play an important role as an endogenous inducer of IFN-gamma expression, thereby contributing to tumor regression.     

Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris.

Crude extracts of four Chinese herbs, Arctium lappa, Astragalus membranaceus, Andrographis paniculata, and Prunella vulgaris, were assessed in several tissue culture lines for anti-HIV activity and for cytotoxicity. One extract, obtained from P. vulgaris, was able to significantly inhibit HIV-1 replication with relatively low cytotoxicity. The active factor was purified using sequential precipitations with ethanol and n-butanol, followed by reverse-phase and gel permeation high-performance liquid chromatographic separations. The active component was anionic with a molecular weight of approximately 10 kDa. The purified extract inhibited HIV-1 replication in the lymphoid cell line MT-4, in the monocytoid cell line U937, and in peripheral blood mononuclear cells at effective concentrations of 6, 30, and 12.5 micrograms/ml, respectively. Pretreatment of uninfected cells with the extract prior to viral exposure did not prevent HIV-1 infection. By contrast, preincubation of HIV-1 with the purified extract dramatically decreased infectiousness. The purified extract was also able to block cell-to-cell transmission of HIV-1, prevented syncytium formation, and interfered with the ability of both HIV-1 and purified gp120 to bind to CD4. PCR analysis confirmed the absence of HIV-1 proviral DNA in cells exposed to virus in the presence of the extract. These results suggest that the purified extract antagonizes HIV-1 infection of susceptible cells by preventing viral attachment to the CD4 receptor.     

Clamp loading,unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human,E. coli and T4 replicases

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nm (K_d ≈ 21 nm ). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t_1/2 ≈ 24 min). β is more stable in solution (K_d < 60 pm ) and on DNA (t_1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (K_d ≈ 250 nm ) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.     

Predicted Near-Future Oceanic Warming Enhances Mercury Toxicity in Marine Copepods

Bulletin of Environmental Contamination and Toxicology - The effects of acute mercury exposure (118&nbsp;µg/L) on the marine copepod Tigriopus japonicus were examined at 22 and...     

A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis

Advances in techniques of molecular biology have made possible the amplification of specific genes from single cells. This has a major clinical application in preimplantation diagnosis of monogenic disorders. However, the incidence of allele specific amplification failure (allele drop out) in heterozygous single cells can lead to misdiagnosis and the transfer of affected embryos. Few studies have been done to investigate the actual cause of allele drop out, although some investigators have succeeded in reducing but not eliminating it. Here we report the efficiency of amplifying both alleles in heterozygous cells lysed according to two different protocols. A total of 177 heterozygous cells from carriers of cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different lysis buffers. Interestingly none of the cells that were lysed with sodium dodecyl sulphate/proteinase K showed any example of allele specific amplification failure whereas in those lysed by KOH/dithiothreitol it was present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results suggest that the phenomenon of allele specific amplification failure is at least in part dependent on the lysis buffer used.      

小鼠补体受体Ⅱ型基因改造后感染EB病毒

通过对小鼠补体受体Ⅱ型基因（CR2）进行定点突变,然后将野生型和突变型小鼠CR2/1（MCR2/1）及人CR2（hCR2)用电穿孔方法导入小鼠SP2/0进行表达,采用免疫组织化学等方法鉴定阳性细胞系。用EB病毒对转染阳性细胞进行感染,EBER-1杂交结果显示,仅转染hCR2和突变型MCR2（mtMCR2)的SP2/0细胞感染了EB病毒,前者感染EB病毒的阳性率较后者高很多。这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。     

基因芯片对PMA激活的血管内皮细胞早期反应基因的研究

目的 :用基因芯片研究 PMA激活的血管内皮细胞的早期反应基因 (imm ediate early response gene,ERG)。方法 :以包含 40 96条人类基因的 DNA芯片检测血管内皮细胞受代谢增强剂 PMA(phorbol myristate acetate)激活后早期的基因表达谱 ,并从中筛查出 ERG。结果 :血管内皮细胞受 PMA作用 6 h后 ,17条基因上调 ,11条下调。数据处理聚类分析表明 17条上调基因中多数 (13/ 17)属蛋白质磷酸酶和转录调控因子基因 ,而下调基因中多数 (9/ 11)为细胞分裂相关基因。结论 :证明PMA激活的人血管内皮细胞早期反应基因主要是转录调控因子和蛋白质磷酸酶基因。