| Part OneResearch on the apoptotic effect and cell cycle arrest by Toxoplasma gondii rhoptry protein16Toxoplasma gondii is an obligate intracellular parasite which can infect almost all of the warm-blooded animals. In recent years, as urban development, population increase and pets team continues to expand, coupled to ignore factors such as food hygiene, infected with Toxoplasma gondii a potential risk of extreme rise. Pregnant women infected with Toxoplasma gondii, expecially during the first three months, usually can give this infection to her fetus, if the infection occurs early in embryonic development can lead to mental retardation, eye and brain disease, multiple organ disease and other serious consequences, affecting the development of the fetus, causing miscarriage, fetal malformations, stillbirth, premature birth, birth defects and other congenital toxoplasmosis. One survey conducted in recent years has shown that China’s congenital toxoplasma infection rates reach as high as22%in birth defects in children, the mentally handicapped and the incidence of psychiatric patients may be bound up with toxoplasma infection.In the process of Toxoplasma gondii invasion into the host cells, rhoptry proteins play an important role. So far, only two Toxoplasma gondii rhoptry proteins have been found to be capable of invading the host cell nucleus, i.e., rhoptry protein16(ROP16) and PP2C-hn. ROP16can quickly invade to the host cell nucleus, it is concerned with virulence of Toxoplasma gondii. It has been proven that ROP16can affect host cell signal transduction and activator of transcription factor (STAT) signaling pathway and thus interfere with the host cell proliferation, differentiation, and apoptosis. However, the specific function of the protein into the nucleus play is not known.Our hypothesis are deal with①since the ROP16can invade into the host nucleus, it can affect the normal physiological functions of the host cell and the nucleus;②since the tropism of Toxoplasma gondii on brain cells, then the intrusion Toxoplasma of the brain cells can affect the normal physiological function of proliferation, differentiation, and apoptosis in these cells, likely, ROP16invade into the nucleus, then it could affect the nucleus function.③Since T. gondii infection of brain cells, it may affect early brain development. We aim to proceed from the ROP16physiological function of basic research to explore change of gene expression profile induced by overexpression of ROP16and the protein interaction with the human body to obtain the downstream target gene information to look for factors that are associated with growth and development, and ROP16and interaction of protein binding to its function impact, reveal the formation of toxoplasmosis and Toxoplasma infection can cause birth defects and mental retardation mechanism.In this paper, the studies will focus on the gene expression prolfile change and confirming the interaction between ROP16and nuclear protein in the body, the main contents and results are as follows:(1) Using cDNA Microarray, we transfected SH-SY5Y with recombinant plasimed ROP16and detect the expression profile changes to screen differentially expressed genes. Microarray analysis had found that819genes were up-regulated and973genes down regulated. GO analysis showed that these genes were involved in several biological processes, including nervous system development, organ formation and proliferation, apoptosis and differentiation.(2) Mass spectrometry had found many human interaction proteins with ROP16: Co-immunoprecipitation assays demonstrated the interaction between ROP16and p53. Confocal microscopy confirmed the co-localization of ROP16with p53in the host nucleus.(3) ROP16affects apoptosis-related genes expression:To validate the microarray results, ROP16was over-expressed in neuroblastoma cells (SH-SY5Y) and real-time PCR was used to detect the expression of apoptosis-related genes. The results found that ROP16can significantly improve the expression of p53, BAX, Caspase-9and inhibit the expression of Bcl-2, thereby promoting apoptosis.(4) The effect of ROP16after its interaction with p53in SH-SY5Y:we tested the impact of the ROP16protein expression on neuronal apoptosis and cell cycle with DNA ladder and flow cytometry. The results had proved that ROP16can promote apoptosis, causing cell cycle arrest. CCK-8experiments confirmed the ROP16inhibit the proliferation of SH-SY5Y cells.(5) The effect of ROP16on p53expression and its phosphorylation at different sites:ROP16can promote the expression of p53protein expression, and improve the S20phosphorylation level, reduce S37phosphorylation levels. Conclusion:ROP16can affect host cell gene expression profiles. ROP16interacts with p53. ROP16inhibits the growth of SH-SY5Y cells. ROP16promotes apoptosis of SH-SY5Y cells and induces cell cycle arrest. ROP16promotes SH-SY5Y apoptosis and cell cycle arrest by increaseing the expression of p53and changes the phosphorylation levels at Ser20/37. Part Two:Research on the molluscicidal targets of ginkgolic acidOncomelania hupensis is a class in Mollusca (Phylum Mollusca), gastropods (Class Gastropoda), based on morphological characteristics, snails genus is divided into two kinds:Hubei snails(Oncomelania hupensis) and tiny snails(Oncomelania minima). Hupensis was domestically distributed in Zhejiang, Jiangsu, Anhui, Jiangxi, Hubei, Hunan, Guangdong and Guangxi. Schistosomiasis is one of the world’s top ten tropical diseases, global infection schistosomiasis is about200million people, nearly600million people are threatened, there are76countries affected, of which the vast majority of developing countries, the disease has seriously jeopardized people’s health and economic development of these countries. Japan schistosomiasis is also one of the most serious epidemics of our country, lake-endemic areas, such as Hunan, Hubei, Jiangxi, Anhui and Jiangsu, accounting for the vast majority of popular area, the national total of435schistosomiasis counties (districts, cities). Although China’s large-scale prevention and control of epidemic has been effectively controlled and made remarkable achievements, but in recent years, due to the impact of floods, population movements, and many other factors, schistosomiasis began to rebound, local transmission of the epidemic, new affected areas increase and the signs indicate that the urban sprawl of schistosomiasis prevent a very daunting task. Therefore, prevention and treatment of schistosomiasis has become one of the strategic objectives of sustainable development.In a previous study, we have confirmed that ginkgo acid has a good molluscides activity. Ginkgo acid treatment can significantly inhibit the speed of the snail’s climbing up, suggesting ginkgo acid can be used as an excellent molluscicide. This effect of inhibiting climbing may be due to the educed ability of energy supply and its mechanism of action is inevitable involved in energy production. This study is a further continuation and broadening of the preliminary work, mainly by transisson electron microscopy and real time PCR, we aim to study the ginkgo acid molluscides on mitochondrial morphological changes and mitochondria related gene expression, respectively. The main contents and results are as follows:(1) Synthesis and purification of the ginkgolic acid monomer(2) Ginkgolic acid treatment of snails can inhibit its climb rate.(3) Transmission electron microscopy showed that can destroy the snails’ mitochondrial structure.(4) Real time PCR confirmed that GA-C13:0can inhibit mitochondrial related genes expression.Conclusion:The snail’s mitochondria might be potential drug targets of the ginkgolic acid and its derivatives.
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