Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Network analysis of skin tumor progression identifies a rewired genetic architecture affecting inflammation and tumor susceptibility

Background  

Germline polymorphisms can influence gene expression networks in normal mammalian tissues and can affect disease susceptibility. We and others have shown that analysis of this genetic architecture can identify single genes and whole pathways that influence complex traits, including inflammation and cancer susceptibility. Whether germline variants affect gene expression in tumors that have undergone somatic alterations, and the extent to which these variants influence tumor progression, is unknown.     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.     

Protopathic stimulant use among children with symptoms of ADHD

The purpose of the current study was to examine protopathic stimulant use among children with the symptoms of ADHD but do not have a diagnosis of ADHD. Protopathic or prodromal stimulant use refers to the use of stimulants by children with the symptoms of ADHD prior to a diagnosis of ADHD. In the current study, we examined children with the symptoms of ADHD who received stimulant treatment across time and with respect to several background variables. Our results indicate that these children who receive stimulant treatment without a diagnosis of ADHD are significantly more like to be eventually diagnosed with ADHD than not. Results also indicate that these children who receive stimulant treatment but do not yet have a diagnosis of ADHD are significantly more likely to have insurance that does not pay for diagnostic procedures. These results are discussed in view of treatment.     

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2.ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.     

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.     

Synthetic Triterpenoids Target the Arp2/3 Complex and Inhibit Branched Actin Polymerization

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.     

The host defense peptide LL‐37 activates the tumor‐suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells

The human cathelicidin LL‐37, a pleiotropic host defense peptide, is down‐regulated in gastric adenocarcinomas. We therefore investigated whether this peptide suppresses gastric cancer growth. LL‐37 lowered gastric cancer cell proliferation and delayed G₁‐S transition in vitro and inhibits the growth of gastric cancer xenograft in vivo. In this connection, LL‐37 increased the tumor‐suppressing bone morphogenetic protein (BMP) signaling, manifested as an increase in BMP4 expression and the subsequent Smad1/5 phosphorylation and the induction of p21^Waf1/Cip1. The anti‐mitogenic effect, Smad1/5 phosphorylation, and p21^Waf1/Cip1 up‐regulation induced by LL‐37 were reversed by the knockdown of BMP receptor II. The activation of BMP signaling was paralleled by the inhibition of chymotrypsin‐like and caspase‐like activity of proteasome. In this regard, proteasome inhibitor MG‐132 mimicked the effect of LL‐37 by up‐regulating BMP4 expression and Smad1/5 phosphorylation. Further analysis of clinical samples revealed that LL‐37 and p21^Waf1/Cip1 mRNA expressions were both down‐regulated in gastric cancer tissues and their expressions were positively correlated. Collectively, we describe for the first time that LL‐37 inhibits gastric cancer cell proliferation through activation of BMP signaling via a proteasome‐dependent mechanism. This unique biological activity may open up novel therapeutic avenue for the treatment of gastric cancer. J. Cell. Physiol. 223: 178–186, 2010. © 2010 Wiley‐Liss, Inc.