Development of pGEMINI, a Plant Gateway Destination Vector Allowing the Simultaneous Integration of Two cDNA via a Single LR-Clonase Reaction

Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector “pGEMINI” represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures

Small molecule receptor tyrosine kinase inhibitor of platelet-derived growth factor signaling (SU9518) modifies radiation response in fibroblasts and endothelial cells

BACKGROUND: Several small receptor tyrosine kinase inhibitors (RTKI) have entered clinical cancer trials alone and in combination with radiotherapy or chemotherapy. The inhibitory spectrum of these compounds is often not restricted to a single target. For example Imatinib/Gleevec (primarily a bcr/abl kinase inhibitor) or SU11248 (mainly a VEGFR inhibitor) are also potent inhibitors of PDGFR and other kinases. We showed previously that PDGF signaling inhibition attenuates radiation-induced lung fibrosis in a mouse model. Here we investigate effects of SU9518, a PDGFR inhibitor combined with ionizing radiation in human primary fibroblasts and endothelial cells in vitro, with a view on utilizing RTKI for antifibrotic therapy. METHODS: Protein levels of PDGFR-α/-β and phosphorylated PDGFR in fibroblasts were analyzed using western and immunocytochemistry assays. Functional proliferation and clonogenic assays were performed (i) to assess PDGFR-mediated survival and proliferation in fibroblasts and endothelial cells after SU9518 (small molecule inhibitor of PDGF receptor tyrosine kinase); (ii) to test the potency und selectivity of the PDGF RTK inhibitor after stimulation with PDGF isoforms (-AB, -AA, -BB) and VEGF+bFGF. In order to simulate in vivo conditions and to understand the role of radiation-induced paracrine PDGF secretion, co-culture models consisting of fibroblasts and endothelial cells were employed. RESULTS: In fibroblasts, radiation markedly activated PDGF signaling as detected by enhanced PDGFR phosphorylation which was potently inhibited by SU9518. In fibroblast clonogenic assay, SU9518 reduced PDGF stimulated fibroblast survival by 57%. Likewise, SU9518 potently inhibited fibroblast and endothelial cell proliferation. In the co-culture model, radiation of endothelial cells and fibroblast cells substantially stimulated proliferation of non irradiated fibroblasts and vice versa. Importantly, the RTK inhibitor significantly inhibited this paracrine radiation-induced fibroblast and endothelial cell activation. CONCLUSION: Radiation-induced autocrine and paracrine PDGF signaling plays an important role in fibroblast and endothelial cell proliferation. SU9518, a PDGFR tyrosine kinase inhibitor, reduces radiation-induced fibroblast and endothelial cell activation. This may explain therapeutic anticancer effects of Imatinib/Gleevec, and at the same time it could open a way of attenuating radiation-induced fibrosis

Stimulating photosynthetic processes increases productivity and water use efficiency in the field

Previous studies have demonstrated that independent stimulation of either electron transport or RuBP regeneration can increase the rate of photosynthetic carbon assimilation and plant biomass. In this paper, we present evidence that a multi-gene approach to simultaneously manipulate these two processes provides a further stimulation of photosynthesis. We report on the introduction of the cyanobacterial bifunctional enzyme fructose-1, 6- bisphosphatase/sedoheptulose-1,7-bisphosphatase or overexpression of the plant enzyme sedoheptulose-1,7-bisphosphatase, together with expression of the red algal protein cytochrome c6, and show that a further increase in biomass accumulation under both glasshouse and field conditions can be achieved. Furthermore, we provide evidence that stimulation of both electron transport and RuBP regeneration can lead to enhanced intrinsic water use efficiency under field conditions

Losartan Slows Pancreatic Tumor Progression and Extends Survival of SPARC-Null Mice by Abrogating Aberrant TGFβ Activation

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation

Linking Employee Stakeholders to Environmental Performance: The Role of Proactive Environmental Strategies and Shared Vision

Drawing on the natural-resource-based view (NRBV), we propose that employee stakeholder integration is linked to environmental performance through firms’ proactive environmental strategies, and that this link is contingent on shared vision. We tested our model with a cross-country and multi-industry sample. In support of our theory, results revealed that firms’ proactive environmental strategies translated employee stakeholder integration into environmental performance. This relationship was pronounced for high levels of shared vision. Our findings demonstrate that shared vision represents a key condition for advancing the corporate greening agenda through proactive environmental strategies. We discuss implications for the CSR and the environmental management literatures, with a particular focus on the NRBV and stakeholder integration debates

SPARC: a matricellular regulator of tumorigenesis

Although many clinical studies have found a correlation of SPARC expression with malignant progression and patient survival, the mechanisms for SPARC function in tumorigenesis and metastasis remain elusive. The activity of SPARC is context- and cell-type-dependent, which is highlighted by the fact that SPARC has shown seemingly contradictory effects on tumor progression in both clinical correlative studies and in animal models. The capacity of SPARC to dictate tumorigenic phenotype has been attributed to its effects on the bioavailability and signaling of integrins and growth factors/chemokines. These molecular pathways contribute to many physiological events affecting malignant progression, including extracellular matrix remodeling, angiogenesis, immune modulation and metastasis. Given that SPARC is credited with such varied activities, this review presents a comprehensive account of the divergent effects of SPARC in human cancers and mouse models, as well as a description of the potential mechanisms by which SPARC mediates these effects. We aim to provide insight into how a matricellular protein such as SPARC might generate paradoxical, yet relevant, tumor outcomes in order to unify an apparently incongruent collection of scientific literature

High-spin states in ¹⁰⁷Pd, ¹⁰⁸Pd, and ¹⁰⁹Ag

High-spin states in 107,108Pd and 109Ag have been studied by charged-particle-γ, γ-γ, and charged-particle-γ-γ coincidence measurements of heavy-ion fusion-evaporation reactions. The high-spin level schemes of these nuclides have been significantly extended and new deformed rotational bands have been found in each nuclide. These bands exhibit the general property of back bends corresponding to the rotational alignment of a pair of h11/2 quasineutrons. Band properties are compared with predictions of the cranked shell model and total Routhian surface calculations. The properties of the yrast and near-yrast bands of all three nuclides are generally consistent with predictions of moderate (β2 = 0.2) prolate deformation