Antiangiogenic and antitumor activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.     

Organic Anion Transporting Polypeptides of the OATP/SLCO Superfamily: Identification of New Members in Nonmammalian Species, Comparative Modeling and a Potential Transport Mode

Organic anion-transporting polypeptides (human, OATPs; other animals, Oatps; gene symbol, SLCO/Slco) form a transport protein superfamily that mediates the translocation of amphipathic substrates across the plasma membrane of animal cells. So far, OATPs/Oatps have been identified in human, rat and mouse tissues. In this study, we used bioinformatic tools to detect new members of the OATP/SLCO superfamily in nonmammalian species and to build models for the three-dimensional structure of OATPs/Oatps. New OATP/SLCO superfamily members, some of which form distinct novel families, were identified in chicken, zebrafish, frog, fruit fly and worm species. The lack of OATP/SLCO superfamily members in plants, yeast and bacteria suggests the emergence of an ancient Oatp protein in an early ancestor of the animal kingdom. Structural models were generated for the representative members OATP1B3 and OATP2B1 based on the known structures of the major facilitator superfamily of transport proteins. A model was also built for the large extracellular region between transmembrane helices 9 and 10, following the identification of a novel homology with the Kazal-type serine protease inhibitors. Along with the electrostatic potential and the conservation of key amino acid residues, we propose a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rocker-switch type of mechanism. Several amino acid residues were identified that may play crucial roles in the proposed transport mechanism. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Chain length effects of oligo(L-lysine)-shelled dendrimers on interaction with DNA

The interaction of novel oligo(L-lysine)-shelled dendrimers (G3-PLL) with DNA was studied by means of circular dichroism spectroscopy, dynamic light scattering, and melting behavior of double-stranded DNA. G3-PLLs having various oligo(L-lysine) (PLL) segment (n = 5-40) were successfully synthesized by graft-polymerization of L-lysine NCA initiated with amino groups at the 3rd-generation poly(amidoamine) dendrimer surface. The ionization property of the newly prepared G3-PLLs were first examined. The evaluated pK(a) values (8.3-8.5) for G3-PLLs were found to be significantly lower than those (9.2-9.4) for the corresponding linear PLLs, probably due to alignment of PLL segments on the three-dimensional core surface. The binding experiments of G3-PLLs to DNA showed that double-stranded DNAs were fairly strongly bound to G3-PLLs primarily through electrostatic interactions. In addition, G3-PLLs served as a DNA cross-linker. A longer PLL-containing G3-PLL was found to interact with DNA more effectively than a shorter one.     

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.     

Gastric mucosal protection against ethanol by EP2 and EP4 signaling through the inhibition of leukotriene C4 production

Prostaglandin (PG)E derivatives are widely used for treating gastric mucosal injury. PGE receptors are classified into four subtypes, EP(1), EP(2), EP(3), and EP(4). We have tested which EP receptor subtypes participate in gastric mucosal protection against ethanol-induced gastric mucosal injury and clarified the mechanisms of such protection. The gastric mucosa of anesthetized rats was perfused at 2 ml/min with physiological saline, agonists for EP(1), EP(2), EP(3), and EP(4), or 50% ethanol, using a constant-rate pump connected to a cannula placed in the esophagus. The gastric microcirculation of the mucosal base of anesthetized rats was observed by transillumination through a window made by removal of the adventitia and muscularis externa. PGE(2) and subtype-specific EP agonists were applied to the muscularis mucosae at the window. Application of 50% ethanol dilated the mucosal arterioles and constricted the collecting venules. Collecting venule constriction by ethanol was completely inhibited by PGE(2) and by EP(2) and EP(4) agonists (100 nM) but not by an EP(1) or an EP(3) agonist. Ethanol-induced mucosal injury was also inhibited by EP(2) and EP(4) agonists. When leukotriene (LT)C(4) levels in the perfusate of the gastric mucosa were determined by ELISA, intragastric ethanol administration elevated the LTC(4) levels sixfold from the basal levels. These elevated levels were significantly (60%) reduced by both EP(2) and EP(4) agonists but not by other EP agonists. Since LTC(4) application at the window constricted collecting venules strongly, and an LTC antagonist reduced ethanol-induced mucosal injury, reductions in LTC(4) generation in response to EP(2) and EP(4) receptor signaling may be relevant to the protective action of PGE(2). The present results indicate that EP(2) and EP(4) receptor signaling inhibits ethanol-induced gastric mucosal injury through cancellation of collecting venule constriction by reducing LTC(4) production.