TH2 cytokines modulate the IL-9R expression on human neutrophils

Interleukin (IL)-9 is associated with key pathological features of asthma such as airway hyperresponsiveness, bronchoconstriction and mucus production. Inflammatory responses mediated by IL-9 rely on the expression of the IL-9R which has been reported on lung epithelial cells, T lymphocytes and recently on airway granulocyte infiltrates. In this study, we assessed the regulatory and constitutive cell surface expression of the IL-9Rα in unfractionated and purified human neutrophils from atopic asthmatics, atopic non-asthmatics and healthy normal controls. We demonstrate that T_H2 cytokines (IL-4 or IL-13) and granulocyte macrophage-colony stimulating factor (GM-CSF) up-regulated mRNA and cell surface expression levels of the IL-9Rα in primary human and HL-60 differentiated neutrophils. Pharmacological inhibition of NF-κB did not affect T_H2-mediated IL-9Rα expression in human neutrophils although IFN-γ and IL-10 down-regulated IL-9Rα expression when co-incubated with IL-4, IL-13 or GM-CSF. Collectively, our results reveal a regulatory function for IFN-γ and IL-10 on modulating the inducible IL-9Rα expression levels on peripheral blood neutrophils by T_H2 cytokines.     

Comparative anatomy of leaves and stems in some species of the South American genus Chrysolaena (Vernonieae,Asteraceae) and taxonomic implications

Vegetative anatomical features are poorly known in the South American genus Chrysolaena. In this study, leaves and stems of six Chrysolaena species were described and compared morphologically and anatomically using diaphanization, microtome serial sectioning and scanning electron microscopy. The species differed in leaf epidermis, type of stomata, shape of anticlinal walls of epidermal cells, trichome density, and presence or absence in stems of small air spaces in the cortical parenchyma and of druse‐shaped oxalate crystals. Furthermore, glandular trichomes and three types of non‐glandular trichomes with different number of basal cells were identified on leaves and stems. Collectively, these features proved instrumental to discriminate among the six studied species, suggesting that leaves and stems of Chrysolaena can represent a source for taxonomically useful characters. We also discuss anatomical features in relation to the environmental conditions in the species’ habitats.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae

A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernemacarpocapsae. The purified protease had an apparent molecular mass of 30 kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K_m, V_max and k_cat values of 409 μM/min, 0.389 μM/min and 24.9 s⁻¹, respectively. The protease was most active at pH 8.0 and 35 °C, and its proteolytic activity was almost completely reduced after incubation at 75 °C for 30 min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24 h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.     

FRS2 via Fibroblast Growth Factor Receptor 1 Is Required for Platelet-derived Growth Factor Receptor ��-mediated Regulation of Vascular Smooth Muscle Marker Gene Expression

Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity and change from a quiescent contractile phenotype to a proliferative synthetic phenotype during physiological arteriogenesis and pathological conditions such as atherosclerosis and restenosis. Platelet-derived growth factor (PDGF)-BB is a potent inducer of the VSMC synthetic phenotype; however, much less is known about the role of fibroblast growth factor-2 (FGF2) in this process. Here, we show using signal transduction mutants of FGF receptor 1 (FGFR1) expressed in rat VSMC that the adaptor protein FRS2 is essential for FGFR1-mediated phenotypic modulation and down-regulation of VSMC smooth muscle α-actin (SMA) gene expression. In addition, we show that PDGF-BB and FGF2 act synergistically to induce cell proliferation and down-regulate SMA and SM22α in VSMC. Furthermore, we show that PDGF-BB induces tyrosine phosphorylation of FGFR1 and that this phosphorylation is mediated by PDGF receptor-β (PDGFRβ), but not c-Src. We demonstrate that FRS2 co-immunoprecipitates with PDGFRβ in a complex that requires FGFR1 and that both the extracellular and the intracellular domains of FGFR1 are required for association with PDGFRβ, whereas the cytoplasmic domain of FGFR1 is required for FRS2 association with the FGFR1-PDGFRβ complex. Knockdown of FRS2 in VSMC by RNA interference inhibited PDGF-BB-mediated down-regulation of SMA and SM22α without affecting PDGF-BB mediated cell proliferation or ERK activation. Together, these data support the notion that PDGFRβ down-regulates SMA and SM22α through formation of a complex that requires FGFR1 and FRS2 and prove novel insight into VSMC phenotypic plasticity.Phenotypic modulation of vascular smooth muscle cells (VSMC)3 is an important step in the development of several pathophysiological processes including atherosclerosis, restenosis, and vascular remodeling (1, 2). During these processes VSMC change from a contractile phenotype to a synthetic phenotype characterized by increased proliferation, migration, increased extracellular matrix production, and decreased expression of contractile proteins, including smooth muscle α-actin (SMA), SM22α, calponin, and myosin heavy chain. Several growth factors including platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor 2 (FGF2), and thrombin have been implicated in the induction of the synthetic phenotype (3). These growth factors bind cell surface receptors and activate intracellular signaling pathways that result in changes in gene expression and cellular phenotype. Understanding the interactions between these pathways may provide insights into mechanisms of phenotypic modulation of VSMC and provide new targets for therapeutic intervention in vascular disease.Experimental evidence using various in vitro and in vivo models points to a role for FGF-FGFR in the phenotypic modulation of VSMC. FGFs and FGFRs are expressed in VSMC and are up-regulated during vascular injury and in atherosclerotic plaque formation (4–6). Balloon injury of rat arteries led to an increase in FGFR expression in VSMC. The up-regulation of FGF and FGFR suggests that they contribute to the pathogenesis of vascular disease. In support of this hypothesis, administration of anti-FGF2 antibodies and FGFR tyrosine kinase inhibitors results in decreased VSMC proliferation, migration, and attenuated neointimal thickening (7).PDGF-BB binds to PDGFRβ and activates several intracellular signaling pathways including ERK, phosphatidylinositol 3-kinase/Akt, and mammalian target of rapamycin (mTOR) (8). Studies have indicated that PDGF-BB induces the release of FGF2 and activation FGFR1, resulting in sustained ERK activation and proliferation of human VSMC (9). When FGFR1 expression was inhibited by RNA interference, PDGF-BB induced transient but not sustained ERK activation.Binding of FGF2 to FGFR1 activates the ERK and phosphatidylinositol 3-kinase/Akt pathways via the adaptor protein FRS2 (10, 11). Upon FGF2 binding, FGFR1 phosphorylates FRS2 on six tyrosine residues that function as docking sites for the SH2 domain-containing proteins Grb2 and SHP2 (12, 13). Grb2 binds Gab1 leading to activation of phosphatidylinositol 3-kinase/Akt, whereas SHP2 activates the Ras-Raf-ERK pathway. FRS2 binds to FGFR1 via a Val-Thr dipeptide in the juxtamembrane region of FGFR1 (14, 15). Deletion of these two amino acids abrogates binding of FRS2 to FGFR1. To determine the role of FRS2 in FGFR1-mediated VSMC phenotypic modulation and to determine the interaction of PDGFRβ with the FGFR1 signaling pathway, we developed a set of FGFR1 signaling pathway deficient mutants and stably expressed them in rat VSMC. In this study we report that PDGFRβ, FGFR1, and FRS2 form a multi-protein complex that is essential for VSMC phenotypic modulation and that stable knockdown of FRS2 inhibits PDGF-BB-mediated down-regulation of VSMC marker gene expression but not PDGF-BB-mediated VSMC proliferation.