In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid-plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an Nterminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.aphid-plant interaction ͉ saliva ͉ RNAi ͉ electrical penetration graph ͉ immunohistochemistry T he ability, or inability, of an aphid to feed on a plant results from a multifaceted interplay between the feeding systems of the insect and the defense systems of the plant (for recent reviews, from several perspectives, of aphid-plant interactions, see refs.

BackgroundIL-1β is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1β may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1β in colon cancer stem cell (CSC) development.MethodsUsing self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1β on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116.ResultsWe found that IL-1β can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1β-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1β-induced spheres, indicating that there might be a close association between EMT and IL-1β-induced CSC self-renewal. Indeed, IL-1β treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1β-induced EMT and stem cell formation.ConclusionOur findings indicate that IL-1β may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1β and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Abstract-Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress. (Circ Res. 2007;100:712-720.)Key Words: nitric oxide Ⅲ thrombospondin-1 Ⅲ ischemic tissue survival Ⅲ CD47 Ⅲ therapeutics T issue viability requires continuous perfusion, which in turn depends on vascular tone, sufficient intravascular volume, and adequate blood oxygenation. 1-3 The contractile status of arterial smooth muscle is the major determinant of vascular tone, with venous tone playing a lesser role. 4,5 Underperfusion of soft tissues is the leading cause of tissue necrosis and secondary delayed wound healing in surgical patients. 6 The complications incurred can be substantial and life threatening. 7 Complications of inadequate tissue perfusion are multiplied in the elderly and patients with hypertension and diabetes because of the general vasculopathies associated with these disease processes. 8,9 Current therapies to improve vascular perfusion combine surgical vessel manipulation/bypass with vasodilators that relax vascular smooth muscle cells (VSMCs). 10,11 The bioactive gas NO is a potent vasodilator 12 that activates soluble guanylate cyclase. The increased cGMP activates cGMPdependent protein kinases and thereby decreases VSMC sensitivity to intracellular Ca 2ϩ , leading to relaxation of contractile proteins. [13][14][15][16] We recently reported that NO/cGMP signaling in VSMCs and endothelial cells is potently inhibited by the secreted protein thrombospondin-1 (TSP1). [17][18][19] We further showed that endogenous TSP1 limits the ability of NO to increase skeletal muscle perfusion and blood oxygen levels in vivo. 20 Following surgically induced acute ischemia in random dorsal skin flaps, endogenous TSP1 also limits tissue survival and recovery of tissue oxygenation. Ischemic tissue survival could be improved by increasing NO levels using isosorbide dinitrate, but the degree of tissue necrosis in treated wild-type mice remained higher than in TSP1-null mice, which achieved essentially complete flap survival following this treatment.To further improve s...

Objective-Decreased blood flow secondary to peripheral vascular disease underlies a significant number of chronic diseases that account for the majority of morbidity and mortality among the elderly. Blood vessel diameter and blood flow are limited by the matricellular protein thrombospondin-1 (TSP1) through its ability to block responses to the endogenous vasodilator nitric oxide (NO). In this study we investigate the role TSP1 plays in regulating blood flow in the presence of advanced age and atherosclerotic vascular disease. Methods and Results-Mice lacking TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately after injury. Treatment of WT and apolipoprotein E-null mice using therapeutic agents that decrease CD47 or enhance NO levels reverses the deleterious effects of age-and diet-induced vasculopathy and results in significantly increased tissue survival in models of ischemia. Conclusion-With increasing age and diet-induced atherosclerotic vascular disease, TSP1 and its receptor CD47 become more limiting for blood flow and tissue survival after ischemic injury. Drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to aging. Key Words: nitric oxide Ⅲ thrombospondin-1 Ⅲ perfusion Ⅲ ischemia Ⅲ wound healing C omplications from peripheral vascular disease, including coronary artery disease and myocardial infarction, stroke, and ischemic vascular disease affect some 80% of people over the age of 65. The elderly have significant alterations in vascular anatomy including a loss of vascular networks 1 and alterations in vascular response to injury. 2 Age is a recognized risk factor for complications after surgery including delayed and incomplete wound healing and tissue loss and accounts for significant morbidity and mortality in this group. [3][4][5][6] Animal studies have confirmed the impact of aging on wound healing. [7][8][9] Tissue perfusion is regulated through the control of blood vessel diameter, which itself is controlled by the contractile state of vascular smooth muscle cells (VSMCs). Nitric oxide (NO) is a primary and ubiquitous dilator of blood vessels. 10 NO is constitutively produced in blood vessels by endothelial nitric oxide synthase (eNOS). NO activates soluble guanylate cyclase (sGC) leading to cGMP production and vasodilation. In aged vascular cells, 11 animals, and people, both eNOS expression and NO production 12 are decreased.We recently reported that thrombospondin-1 (TSP1) blocks NO-driven VSMC relaxation in a CD47-dependent manner. 13-15 NO-driven alterations in blood flow are substantially greater in the absence of TSP1 or CD47. Given the deleterious effects of aging on the cardiovascular system, we wanted to determine whether blocking of TSP1 inhibition of NO signaling would provide significant tissue protection in senescent animals. Aged WT and apolipoprotein E (apoE)-null mice (with diet-driv...