日本血吸虫铁蛋白基因的克隆、表达及其免疫诊断的研究

目的：构建、表达、纯化日本血吸虫铁蛋白基因的原核表达重组蛋白，并初步观察其免疫诊断价值。方法：将日本血吸虫铁蛋白基因亚克隆于pGMC载体，转化重组体到大肠杆菌E．coli 2566进行表达；采用电泳层析法分离纯化日本血吸虫铁蛋白；用SDS-PAGE和Western blot鉴定表达纯化产物；用Dot-ELISA分别检测了血吸虫病人及正常人的血清。结果：重组的日本血吸虫铁蛋白以GMC(粒细胞巨噬细胞集落刺激因子)．日本血吸虫铁蛋白融合蛋白的形式在细菌中得到高效表达，分子量为40kD，能被日本血吸虫感染兔血清识别。用重组铁蛋白检测30例日本血吸虫病人血清和30例正常人血清，阳性率和假阳性率分别为93．3％和3．3％。结论：成功地表达纯化日本血吸虫重组铁蛋白．且该蛋白具有一定的免疫诊断价值．     

Stage and tissue specific differences in SjBMI1, a Polycomb protein in Schistosoma japonicum

Polycomb group protein BMI1, plays a central role in the stem cell pluripotency and development in metazoans. A gene encoding BMI1 homologue in the Schistosoma japonicum (SjBMI1) was cloned and identified. The deduced amino acid sequence shows high identity to the homologues from Schistosoma mansoni and Homo sapiens. Quantitative real time polymerase chain reaction (RT-PCR) and Western blot analysis revealed that the SjBMI1 is highly expressed in adult worms and eggs, not in cercariae. By immunofluorescent studies, SjBMI1 was localized to testes, ovaries of mixed sex infected adult worms, but not of single sex infected adult worms. The study reveals the SjBMI1 expression profile in developmental stages and localization characteristic and provides a clue that it may be associated with reproductive development of S. japonicum.     

Isolation of Schistosoma japonicum egg-derived neutrophil stimulating factor: its role on eosinophil chemotactic factor release from neutrophils

Neutrophil stimulating factor (NSF), which can stimulate polymorphonuclear neutrophils (PMNs) to release eosinophil chemotactic factor (ECF), was isolated from Schistosoma japonicum soluble egg extract (SEA). The release of ECF from PMNs began as early as 5 min after stimulation and reached a peak at 40 min, and was dependent on the concentration of SEA. After Sephadex G150 gel filtration, toluene extraction, Sephadex G25 gel filtration and Dowex 1 X 8 anion exchange chromatography, NSF was identified as a hydrophilic and negatively charged component with a molecular weight of about 1,000 daltons. It was heat-stable at 100 degrees C for 60 min. NSF was easily separable from SEA-derived neutrophil chemotactic factor or from SEA-derived ECF. PMNs are suggested to be one of the sources of ECF in the eosinophil accumulation of granulomatous lesions around the deposited eggs in schistosomiasis japonica.     

Interferon tau and its immunobiological role in ruminant reproduction

Interferon tau (IFN-tau) is an key cytokine in maintaining pregnancy in ruminants. It is produced by the ruminant conceptus around the time of implantation. IFN-tau belongs to the type I interferon family but, unlike the other members of this group, it is not virus inducible and its expression is temporal and restricted to the trophoblast cells of the ruminant conceptus. The main target of the paracrine action of this cytokine is the endometrium. It changes the prostaglandin metabolism and secretory function of the cells by upregulating the secretion of several proteins. It also presents immunomodulatory action towards leukocytes by changing their proliferative responses and cytokine production. This cytokine activity in reproductive biology and immunology has been intensively explored for the last ten years. It has been regarded as a potential tool in improving the performance and biotechnological processes in ruminant reproduction. Additionally, its high antiviral potency and low cytotoxicity in comparison with IFN-alpha has placed this cytokine in the group of possible therapeutics in human and animal medicine.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells

Oxidant stress-mediated regulation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic outcomes in lung epithelium, yet a role for Ca2+ and Ca2+/cAMP-response element binding protein (CREB) in ERK1/2 signaling has not been defined. In this study, we tested the hypotheses that oxidants induce Ca2+-mediated phosphorylation of ERK and CREB, and that CREB is required for oxidant-induced proliferation and apoptosis. H2O2 initiated an influx of extracellular Ca2+ that was required for phosphorylation of both ERK and CREB in C10 lung epithelial cells. H2O2-mediated CREB phosphorylation was sensitive to MEK inhibition, suggesting that crosstalk between Ca2+, ERK, and CREB signaling pathways contributes to the oxidant-induced response. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos steady-state mRNA levels, but unexpectedly enhanced bcl-2 steady-state mRNA levels after H2O2 exposure. Whereas inhibition of CREB activity had no detectable effect on H2O2 stimulation of cell cycle, loss of CREB activity significantly reduced the number of cells undergoing apoptosis. These data support a novel communication between Ca2+-ERK1/2 and CREB elicited by H2O2, and further provide evidence that CREB is an important regulator of apoptosis in oxidant-mediated responses of lung epithelial cells.     

Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein

Schistosomiasis is a worldwide parasitic disease, and while it can be successfully treated with chemotherapy, this does not prevent reinfection with the parasite. Adenovirus vectors have been widely used for vaccine delivery, and a vaccination approach has the potential to prevent infection with Schistosoma. Here, we developed a recombinant adenoviral vector that expresses Schistosoma japonicum inhibitor apoptosis protein (Ad-SjIAP) and assessed its immunoprotective functions against schistosomiasis in mice. Murine immune responses following vaccination were investigated using enzyme-linked immunosorbent assays (ELISA), lymphocyte proliferation, and cytokine assays. The protective immunity in mice was evaluated by challenging with S. japonicum cercariae. Our results indicated that immunization with the Ad-SjIAP in mice induced a strong serum IgG response against IAP including IgG1, IgG2a, and IgG2b. In addition, lymphocyte proliferation experiments showed that mice treated with Ad-SjIAP significantly increased the lymphocyte response upon stimulation with recombinant Schistosoma japonicum inhibitor apoptosis protein (rSjIAP). Moreover, cytokine assays indicated that vaccination of Ad-SjIAP significantly increased the production of interferon (IFN)-γ and IL-2 as compared to the corresponding control group. Furthermore, following the challenge with S. japonicum cercariae, the vaccine conferred moderate protection, with an average rate of 37.95 % for worm reduction and 31.7 % for egg reduction. Taken together, our preliminarily results suggested that schistosoma IAP may be a potential vaccine against S. japonicum and that adenoviral vectors may serve as an alternative delivery vehicle for schistosome vaccine development.     

Molecular characterization,expression profile,and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum

Parasitology Research - Schistosomes are the causative agents of human schistosomiasis, which is endemic in tropical and subtropical zones. CD63 is a member of the tetraspanin protein family widely...     

Relationship of neuronal vulnerability and calcium binding protein immunoreactivity in ischemia

Summary The relationship between neuronal calcium binding protein content (calbindin D_28K: CaBP and parvalbumin : PV) and vulnerability to ischemia was studied in different regions of the rat brain using the four vessel occlusion model of complete forebrain ischemia. The areas studied, i.e. the hippocampal formation, neocortex, neostriatum and reticular thalamic nucleus (RTN), show a characteristic pattern of CaBP and PV distribution, and are involved in ischemic damage to different degrees. In the hippocampal formation CaBP is present in dentate granule cells and in a subpopulation of the CA1 pyramidal cells, the latter being the most and the former the least vulnerable to ischemia. Non-pyramidal cells containing CaBP in these regions survive ischemia, whereas PV-containing non-pyramidal cells in the CA1 region are occasionally lost. Hilar somatostatin-containing cells and CA3 pyramidal cells contain neither PV nor CaBP. Nevertheless, the latter are resistant to ischemia and the former is the first population of cells that undergoes degeneration. Supragranular pyramidal neurons containing CaBP are the most vulnerable cell group in the sensory neocortex. In the RTN the degenerating neurons contain both PV and CaBP. In the neostriatum, ischemic damage involves both CaBP-positive and negative medium spiny neurons, although the degeneration always starts in the dorsolateral neostriatum containing relatively few CaBP-positive cells. The giant cholinergic interneurons of the striatum contain neither CaBP nor PV, and they are the most resistant cell type in this area. These examples suggest the lack of a consistent and systematic relationship between neuronal CaBP or PV content and ischemic vulnerability. It appears that some populations of cells containing CaBP or PV are more predisposed to ischemic cell death than neurons lacking these proteins. These neurons may express high levels of calcium binding proteins because their normal activity may involve a high rate of calcium uptake and/or intraneuronal release.     

日本血吸虫中国株次黄嘌呤-鸟嘌呤磷酸核糖转移酶真核重组质粒的构建及其动物免疫保护性研究

目的 克隆日本血吸虫次黄嘌呤-鸟嘌呤磷酸核糖转移酶（SjHGPRT）全长编码基因，研究其DNA疫苗诱导小鼠的免疫保护作用。方法 将全长SjHGPRT基因克隆到真核表达载体pcD—NA3上，构建真核表达质粒pcDNA3-SjHGPRT，用经双酶切、PCR和测序鉴定正确的真核表达质粒免疫昆明小鼠3次，每次间隙2周，第3次免疫后第2周进行日本血吸虫尾蚴攻击感染，感染后42d处死小鼠，收集成虫、肝脏和24h粪便，计算减虫率和减卵率。结果 成功构建真核表达质粒pcDNA3-SjHGPRT。动物免疫实验结果显示：pcDNA3-SjHGPRT疫苗组减虫率23．17％、肝减卵率41．64％、粪减卵率40．57％，每雌虫子宫内卵减卵率26．95％，与对照组相比差异均具有统计学意义（P〈0．05，P〈0．01）。每雌虫子宫内卵减卵率及肝、粪减卵率明显高于减虫率，表明pcDNA3-SjHGPRT疫苗可能主要在抗血吸虫卵胚发育或抗生殖方面起作用。结论 日本血吸虫次黄嘌呤-鸟嘌呤磷酸核糖转移酶DNA疫苗可诱导抗血吸虫卵胚发育或抗生殖作用，具有潜在的疫苗研究与开发价值。     

Histidine-rich calcium binding protein,a sarcoplasmic reticulum protein of striated muscle,is also abundant in arteriolar smooth muscle cells

Summary Histidine-rich calcium binding protein (HRC) is a luminal sarcoplasmic reticulum protein abundant in skeletal and cardiac muscle. Using immunofluorescence to examine non-muscle tissues, we now show that HRC is also abundant in the smooth muscle cells lining the walls of small arteries and arterioles. Arterioles that contain only one or two layers of smooth muscle cells are very brightly stained while small muscular arteries demonstrate a lesser degree of immunoreactivity only in cells just adjacent to the lumen of the vessel. In contrast, visceral smooth muscle cells from the gastrointestinal and genitourinary tracts show no HRC immunofluorescence. We also examined the subcellular distribution of HRC in arteriolar smooth muscle by immunoelectron microscopy. HRC was found in electron-dense vesicles beneath the plasma membrane, in small electron-lucent vesicles and in the nuclear envelope, suggesting a location within a calcium-sequestering compartment. These findings suggest that HRC plays a role in sarcoplasmic reticulum function that is unique to striated and arteriolar smooth muscle.