Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%–75% (p = 0.011) and 69%–89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability

Natalizumab affects T-cell phenotype in multiple sclerosis: implications for JCV reactivation

The anti-CD49d monoclonal antibody natalizumab is currently an effective therapy against the relapsing-remitting form of multiple sclerosis (RRMS). Natalizumab therapeutic efficacy is limited by the reactivation of the John Cunningham polyomavirus (JCV) and development of progressive multifocal leukoencephalopathy (PML). To correlate natalizumab-induced phenotypic modifications of peripheral blood T-lymphocytes with JCV reactivation, JCV-specific antibodies (serum), JCV-DNA (blood and urine), CD49d expression and relative abundance of peripheral blood T-lymphocyte subsets were longitudinally assessed in 26 natalizumab-treated RRMS patients. Statistical analyses were performed using GraphPad Prism and R. Natalizumab treatment reduced CD49d expression on memory and effector subsets of peripheral blood T-lymphocytes. Moreover, accumulation of peripheral blood CD8+ memory and effector cells was observed after 12 and 24 months of treatment. CD4+ and CD8+ T-lymphocyte immune-activation was increased after 24 months of treatment. Higher percentages of CD8+ effectors were observed in subjects with detectable JCV-DNA. Natalizumab reduces CD49d expression on CD8+ T-lymphocyte memory and effector subsets, limiting their migration to the central nervous system and determining their accumulation in peripheral blood. Impairment of central nervous system immune surveillance and reactivation of latent JCV, can explain the increased risk of PML development in natalizumab-treated RRMS subjects

Intron Evolution: Testing Hypotheses of Intron Evolution Using the Phylogenomics of Tetraspanins

BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life. METHODOLOGY/PRINCIPAL FINDINGS: We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1-6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions. CONCLUSIONS/SIGNIFICANCE: Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty

Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation

Down-regulation of TM4SF is associated with the metastatic potential of gastric carcinoma TM4SF members in gastric carcinoma

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to clarify the clinical significance of TM4SF members CD9, CD63 and CD82 in human gastric carcinoma.</p> <p>Methods</p> <p>By employing RT-PCR and immunohistochemistry, we studied the expression of CD9, CD63 and CD82 in 49 paired tissue specimens of normal gastric mucosa and carcinoma. All tissues were obtained from patients who underwent curative surgery.</p> <p>Results</p> <p>All normal gastric epithelium and gastric ulcer tissues strongly expressed transcripts and proteins of CD9, CD63 and CD82 as compared with corresponding controls. We found a significant correlation between CD63 mRNA level and different pM statuses (P = 0.036). Carcinomas in M0 stage revealed a stronger expression of CD63 than carcinomas in M1 stage. Expression of CD9 protein was found significantly stronger in pN0, pM0 than in advanced pN stages (P = 0.03), pM1 (P = 0.013), respectively. We found the relationship between CD63 expression, gender (p = 0.09) and nodal status (p = 0.028), respectively. Additionally, advanced and metastasized tumor tissues revealed significantly down-regulated CD82 protein expression (p = 0.033 and p = 0, respectively), which correlated with the tumor pTNM stage (p = 0.001).</p> <p>Conclusion</p> <p>The reduction of CD9, CD63 and CD82 expression are indicators for the metastatic potential of gastric carcinoma cells. Unlike their expression in other tumor types, the constitutive expression of CD63 may indicate that this factor does play a direct role in human gastric carcinogenesis.</p

Epithelial Membrane Protein-2 Promotes Endometrial Tumor Formation through Activation of FAK and Src

Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

BACKGROUND: EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. CONCLUSION: EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs

The Tetraspanins CD9 and CD81 Regulate CD9P1-Induced Effects on Cell Migration

CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a β1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin α2β1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9(hi), SSEA-1(−) Cells in Embryonic Stem Cell-Derived Embryoid Bodies

BACKGROUND: Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(−) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(−) cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs