Transcriptional profile of SH-SY5Y human neuroblastoma cells transfected by Toxoplasma rhoptry protein 16.

Toxoplasma rhoptry protein 16 (ROP16) is crucial in the host-pathogen interaction by acting as a virulent factor during invasion. To improve understanding of the molecular function underlying the effect of ROP16 on host cells, the present study analyzed the transcriptional profile of genes in the ROP16­transfected SH­SY5Y human neuroblastoma cell line. The transcriptional profile of the SH­SY5Y human neuroblastoma cell line overexpressing ROP16 were determined by microarray analysis in order to determine the host neural cell response to the virulent factor. Functional analysis was performed using the Protein Analysis Through Evolutionary Relationships classification system. The ToppGene Suite was used to select candidate genes from the differentially expressed genes. A protein­protein interaction network was constructed using Cytoscape software according to the interaction associations determined using the Search Tool for the Retrieval of Interacting Genes/Proteins. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis of the selected genes confirmed the results of the microarray. The results showed that 383 genes were differentially expressed in response to ROP16 transfection, of which 138 genes were upregulated and 245 genes were downregulated. Functional analysis indicated that the differentially expressed genes (DEGs) were involved in several biological processes, including developmental process, biological regulation and apoptotic process. A total of 15 candidate genes from the DEGs were screened using the ToppGene Suite. No significant differences in expression were observed between the RT­qPCR data and the microarray data. Transfection with ROP16 resulted in alterations of several biological processes, including nervous system development, apoptosis and transcriptional regulation. Several genes, including CXCL12, BAI1, ZIC2, RBMX, RASSF6, MAGE­A6 and HOX, were identified as significant DEGs. Taken together, these results may contribute to understanding the mechanisms underlying the functions of ROP16 and provide scope for further investigation of the pathogenesis of Toxoplasma gondii.

Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of the tissue factor (TF)-initiated coagulation pathway. Both circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis. However, the significance of endothelial cell-anchored TFPI in cancer biology remains largely unexplored. We generated mice with full-length disruption of TFPI (including TFPIα and TFPIß isoforms) in endothelial cells, using a Cre-LoxP system and gene inactivation (GI) strategy. Experimental pulmonary tumor metastasis models were used with TFPI-deficient mice to evaluate the role of endothelial cell-anchored TFPI in cancer progression. Finally, lung microvascular permeability and microenvironment were investigated. TFPI-deficient mice were viable and fertile, and showed decreased plasma TFPI levels and lung TFPI levels as compared with their control littermates. TFPI deficiency in endothelial cells promoted pulmonary tumor metastasis with an increased vascular permeability and altered lung microenvironment. Our observations suggest that endothelial cell-anchored TFPI controls lung tumor metastasis, and does so largely through the inhibition of local TF-induced thrombin generation and the regulation of the lung microenvironment in mice.

Toxoplasma gondii Rhoptry Protein ROP16 Mediates Partially SH-SY5Y Cells Apoptosis and Cell Cycle Arrest by Directing Ser15/37 Phosphorylation of p53.

Toxoplasma rhoptries, an unusual set of apical organelles that are associated with Toxoplasma infection may cause subversion of the host cell functions. Parasite rhoptry protein 16 (ROP16) is a regulator of host cell transcription during cell invasion in which it migrates into the host cell cytoplasm and subsequently localizes to the nucleus. In the present study, we found that overexpression of ROP16 could partially mediate human neuroblastoma SH-SY5Y apoptosis (12.47%) and cell cycle arrest in G1 phase (60.77%) in a p53 dependent manner by influencing the expression of Bax/Bcl-2 and p21/CDKs. ROP16 was identified to co-localize with p53, a novel direct interaction partner in the nucleus of SH-SY5Y. Furthermore, SH-SY5Y apoptosis via the mitochondria-dependent p53 pathway and cell cycle arrest caused by ROP16 dealt with direct serine 15/37 phosphorylation of p53. Our studies provide a new mechanism by which ROP16 interacts with the nucleus proteins which subsequently subverts the host cells functions.

Tissue factor pathway inhibitor-2: a novel gene involved in zebrafish central nervous system development.

Tissue factor pathway inhibitor-2 (Tfpi-2) is an important serine protease inhibitor in the extracellular matrix (ECM), but its precise physiological significance remains unknown. This work is part of a series of studies intended to investigate functional roles of Tfpi-2 and explore the underlying molecular mechanisms. First, we cloned and identified zebrafish Tfpi-2 (zTfpi-2) as an evolutionarily conserved protein essential for zebrafish development. We also demonstrated that ztfpi-2 is mainly expressed in the central nervous system (CNS) of zebrafish, and embryonic depletion of ztfpi-2 caused severe CNS defects. In addition, changes of neural markers, including pax2a, egr2b, huC, ngn1, gfap and olig2, confirmed the presence of developmental abnormalities in the relevant regions of ztfpi-2 morphants. Using microarray analysis, we found that members of the Notch pathway, especially her4 and mib, which mediate lateral inhibition in CNS development, were also downregulated. Intriguingly, both her4 and mib were able to partially rescue the ztfpi-2 morphant phenotype. Furthermore, Morpholino knockdown of ztfpi-2 resulted in upregulation of neuronal markers while downregulation of glial markers, providing evidence that the Notch pathway is probably involved in ztfpi-2-mediated CNS development.

The interaction of the second Kunitz-type domain (KD2) of TFPI-2 with a novel interaction partner, prosaposin, mediates the inhibition of the invasion and migration of human fibrosarcoma cells.

TFPI-2 (tissue factor pathway inhibitor-2) has recently been recognized as a new tumour suppressor gene. Low expression of this protein in several types of cancers allows for enhanced tumour growth, invasion and metastasis. To investigate the molecular mechanism responsible for the tumour-suppressor effects of TFPI-2, we performed yeast two-hybrid analysis and identified PSAP (prosaposin) as a TFPI-2-interacting partner. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. The region of TFPI-2 that interacts with PSAP is located in the KD2 (Kunitz-type domain 2). Further study showed that PSAP does not affect the function of TFPI-2 as a serine proteinase inhibitor, but that TFPI-2 could inhibit the invasion-promoting effects of PSAP in human HT1080 fibrosarcoma cells. The results of the present study revealed that TFPI-2 interacts with PSAP, which may play an important role in the physiology and pathology of diseases such as cancer.

[Preparation of five ginkgolic acid monomers and their molluscicidal effects against Oncomelania hupensis].

OBJECTIVE: To investigate the molluscicidal activities of the ginkgolic acid(GA) monomers isolated and purified from GAs. METHODS: Five monomers of GAs from the sarcotesta of Ginkgo biloba. were extracted by petrol ether, separated by silica gel column chromatography, purified by semi-prepared reversed-phased HPLC, and identified by LC-MS analysis. The molluscicidal activities of GAs and their monomers against Oncomelania hupensis were determined as referring to the WHO guidelines for laboratory molluscicidal test. RESULTS: The five purified ginkgolic acid monomers were GA(13:0), GA(15:0), GA(15:1), GA(17:1) and GA(17:2), with a side chain of 13, 15, 17 alkyl or ethylenic radicals res pectively on their benzene loop. The five monomer proportions to the total GAs were 17.6%, 3.2%, 52.3%, 23.3% and 3.6% respectively. The order of molluscicidal activities for the five monomers was as follows: GA(13:0)>GA(15:1)>GA(15:0)>GA(17:1)>GA(17:2), and their LC50 for snails was 20.79 mg/L, 22.28 mg/L, 33.76 mg/L, 51.89 mg/L, and 59.10 mg/L respectively after immersion for 24 hours. Two monomers, GA(13:0), and GA(15:1) inhibited the snails' climbing up significantly. CONCLUSION: The molluscicidal activities of GAs may be dependent on the monomer's structure with different number of carbon molecules and double-bonds on the side carbon-chain. The two monomers, GA(13:0) and GA(15:1), are mainly responsible for the molluscicidal activities of GAs and both effectively inhibit snails' climbing up as well. GA(15:0) also shows certain molluscicidal activity.

[Preliminary survey on concentration of indoor allergens derived from dust mites in residential homes in Shanghai].

OBJECTIVE: To investigate the level of indoor dust mite allergens in residential homes in Shanghai. METHODS: The allergenic proteins, Dff F3 and Der f III (well known allergen) from the whole culture extract of Dermatophagoides farinae were purified by chromatography and sedimentation techniques. Allergic activities of both proteins were identified by skin prick test and RAST. After preparation of specific IgG anti-Dff F3, anti-Der f III, and extraction of indoor allergens from 200 indoor dust samples collected from residential homes, the allergen concentration was measured by sandwich ELISA. Allergen level was expressed in geometric mean and range, the analysis of variance (ANOV) was used to determine the level of significance between groups. RESULTS: Dff F3 and Der f III were demonstrated strongly allergic activities, which can be highly recognized with IgE from sera of the mite-allergic patients with asthma. In the sampled 200 homes, the proportion of homes with Dff F3 level of >10 microg/g was 57.0%, 29.5% for group of 2-10 microg/g, and 13.5% for group of <2 microg/g. The proportion of homes with Der f III level of >10 microg/g was 53.5%, 32.0% for group of 2-10 microg/g, 14.5% for group of <2 microg/g. CONCLUSION: Selected residential homes in Shanghai were found more likely to have high level of dust mite allergens. Dff F3 was identified as the stronger allergic fraction.

Comparative analysis and expression of CLUL1, a cone photoreceptor-specific gene.

PURPOSE: To characterize CLUL1, a cone photoreceptor-specific gene. METHODS: A comparative genomics approach was used to analyze the gene organization and protein sequence of a retinal clusterin-like protein and to identify conserved elements between human and dog. Its expression was studied by Northern and Western analyses and its localization by in situ hybridization and immunocytochemistry. RESULTS: The CLUL1 sequences of the human and dog share 85% and 73% identity, respectively, at the nucleotide and deduced amino acid level. The gene is organized into nine exons and shows strong homology, not only in exonic but also in some intronic sequences between the species. The polypeptide homology of CLUL1 to CLU, a molecular chaperone, indicates structural similarity of the two proteins. However, these data demonstrated that they present different expression profiles in the tissues, in retinal development, and in retinal diseases. Finally, CLUL1 was localized to retinal cone photoreceptor cells and a different immunolabeling in light- and dark-adapted retinas was shown. CONCLUSIONS: CLUL1 represents a potentially important gene and a candidate locus for retinal disease, particularly those diseases that affect cones.