This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.jmolecularsignaling.com The latest articles from Journal of Molecular Signaling (ISSN 1750-2187) published by BioMed Central Background: Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (beta) subunit of casein kinase 2 (CK2beta) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2beta in IVA-infected A549 human epithelial lung cells. Results: Activation of CK2beta was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2beta mRNA to silence CK2beta protein expression in A549 cells without affecting expression of the CK2alpha subunit. CK2beta gene silencing led to increased virus titers, consistent with the inhibition of CK2beta during IVA replication. Notably, virus titers increased significantly when CK2beta siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2beta stimulator. CK2beta absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2beta siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content. Conclusions: This study demonstrates the role of cellular CK2beta protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2beta protein and regulation of IVA replication in infected cells. Overall, the data suggest that expression and activation of CK2beta inhibits IVA replication by regulating the virus entry process and viral protein synthesis. http://www.jmolecularsignaling.com/content/3/1/13 Henju Marjuki, Christoph Scholtissek, Hui-Ling Yen and Robert G Webster Journal of Molecular Signaling 2008, 3:13 2008-07-23 doi:10.1186/1750-2187-3-13 Journal of Molecular Signaling 1750-2187 3 13 2008-07-23 Background: A previous study from this laboratory showed that Galpha12 members participate in the production of inflammatory cytokines. In spite of the identification of B cell homeostasis responses regulated by Galpha13, the functional roles of Galpha12 members in the production of immunoglobulin (Ig) isotypes remained unknown. This study investigated whether Galpha12 members are involved in the Ig isotype antibody production with the purpose of establishing their functions in thymus-dependent and thymus-independent humoral responses. Results: Mice lacking Galpha12 and/or Galpha13 showed an impaired antigen-specific antibody production promoted by challenge(s) of ovalbumin or trinitrophenyl-lipopolysaccharide (TNP-LPS), used for thymus-dependent and thymus-independent stimuli, respectively. Homozygous knockout (KO) of Galpha12 or double heterozygous KO of Galpha12/Galpha13 significantly reduced the antigen-specific total IgG level after multiple ovalbumin immunizations with decreases in the production of IgG1, IgG2a and IgG2b subclasses, as compared to wild type control. In contrast, IgM production was not decreased. Moreover, mice deficient in Galpha12 or partially deficient in Galpha13 or Galhpa12/Galpha13 showed significantly low production of IgG2b in response to TNP-LPS. In TNP-LPS-injected mice, IgG1 and IgG2a productions were unaffected by the G protein KOs. Conclusion: Our results demonstrate that both Galpha12 and Galpha13 are essentially involved in thymus-dependent and independent productions of IgG subclasses, implying that the G-proteins contribute to the process of antigen-specific IgG antibody production. http://www.jmolecularsignaling.com/content/3/1/12 Song Jin Lee, Woo Hyung Lee, Chang Ho Lee and Sang Geon Kim Journal of Molecular Signaling 2008, 3:12 2008-07-12 doi:10.1186/1750-2187-3-12 Journal of Molecular Signaling 1750-2187 3 12 2008-07-12 Background: Understanding the relative importance of signaling pathway components which regulate a specific cellular response is a major focus of current efforts in biology. This interest, along with the inherit complexity of these systems, is driving the development of approaches capable of providing both quantitative predictions as well as guiding the design of future experiments. Of particular interest is the establishment of methods for the analysis of cellular-level input-output signaling relationships that have been characterized over time. Results: Work by the Alliance for Cellular Signaling (AfCS) has provided an extensive profile of ligand-induced changes in protein phosphorylation state and cytokine output response in macrophage-like RAW 264.7 cells. Using model averaging with partial least squares (PLS) or principal components regression (PCR), we compared multivariate models quantitatively predicting cytokine release and identifying key regulatory components of the underlying signaling pathways. We paid particular attention to the effect of metrics extracted from the experimentally derived signaling time courses so as to determine whether the inclusion of such temporal information improved model predictions. Results indicate that we were able to determine the key biological predictors responsible for generating a specific cytokine response, with model R2 values ranging from 0.48 to 0.93. Furthermore, for this data set, the use of time metrics was found to be of mixed value, with increased and/or more appropriate sampling likely being required to improve predictive performance. Conclusion: The use of multivariate approaches and model averaging provides a valuable predictive framework for quantitative studies of these complex biological processes. Results of this work also point to several issues for consideration in the design of similar large-scale interrogations. http://www.jmolecularsignaling.com/content/3/1/11 Yang Wu, Gary L Johnson and Shawn M Gomez Journal of Molecular Signaling 2008, 3:11 2008-05-22 doi:10.1186/1750-2187-3-11 Journal of Molecular Signaling 1750-2187 3 11 2008-05-22 Background: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. Results: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the β-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3β hyperphosphorylation and β-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. Conclusion: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to β-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a. http://www.jmolecularsignaling.com/content/3/1/10 Thecla Constantinou, Fabrizio Baumann, Markus D Lacher, Susanne Saurer, Robert Friis and Arun Dharmarajan Journal of Molecular Signaling 2008, 3:10 2008-05-02 doi:10.1186/1750-2187-3-10 Journal of Molecular Signaling 1750-2187 3 10 2008-05-02 Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation. http://www.jmolecularsignaling.com/content/3/1/9 Shilpa Ramani and Chelliah Jayabaskaran Journal of Molecular Signaling 2008, 3:9 2008-04-25 doi:10.1186/1750-2187-3-9 Journal of Molecular Signaling 1750-2187 3 9 2008-04-25 Background: Vascular endothelial growth factor receptor-2 (VEGFR-2, KDR), a receptor tyrosine kinase, regulates mitogenic, chemotactic, hyperpermeability, and survival signals in vascular endothelial cells in response to its ligand vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF). SHP-1 is a protein tyrosine phosphatase known to negatively regulate signaling from receptors such as EGF receptor, IL3 receptor, erythropoietin receptor and also KDR. However, the mechanism by which SHP-1 executes KDR dephosphorylation, the targeted tyrosine residue(s) of KDR and also overall downstream signaling or phenotypic change(s) caused, is not defined. Results: Here, we have demonstrated that KDR and SHP-1 are constitutively associated and upon VEGF treatment, the phosphatase activity of SHP-1 is stimulated in a c-Src kinase dependent manner. Knockdown of SHP-1 by siRNA or inhibition of c-Src by an inhibitor, results in augmented DNA synthesis perhaps due to increased phosphorylation of at least three tyrosine residues of KDR 996, 1059 and 1175. On the other hand, neither tyrosine residue 951 of KDR nor VEGF-mediated migration is affected by modulation of SHP-1 function. Conclusion: Taken together our results define the tyrosine residues of KDR that are regulated by SHP-1 and also elucidates a novel feed back loop where SHP-1 is activated upon VEGF treatment through c-Src and controls KDR induced DNA synthesis, eventually leading to controlled angiogenesis. http://www.jmolecularsignaling.com/content/3/1/8 Resham Bhattacharya, Junhye Kwon, Enfeng Wang, Priyabrata Mukherjee and Debabrata Mukhopadhyay Journal of Molecular Signaling 2008, 3:8 2008-03-31 doi:10.1186/1750-2187-3-8 Journal of Molecular Signaling 1750-2187 3 8 2008-03-31 Background: We have recently shown that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, inhibits angiogenesis. However, the molecular mechanisms by which EGCG inhibits angiogenesis have never been investigated. In this study, we examined the interaction of PI3K/AKT and MEK/ERK pathways on the regulation of FOXO transcription factors, which ultimately control the antiangiogenic effects of EGCG. Results: Inhibition of PI3K/AKT and MEK/ERK pathways interact synergistically to inhibit migration and capillary tube formation of HUVEC cells and further enhanced the antiangiogenic effects of EGCG. Inhibition of AKT and MEK kinases synergistically induced FOXO transcriptional activity, which was further enhanced in the presence of EGCG. Phosphorylation deficient mutants of FOXO induced FOXO transcriptional activity, inhibited HUVEC cell migration and capillary tube formation. Inhibition of FOXO phosphorylation also enhanced antiangiogenic effects of EGCG through transcriptional activation of FOXO. Conclusion: Inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to regulate antiangiogenic effects of EGCG through activation of FOXO transcription factors. The activation of FOXO transcription factors through inhibition of these two pathways may have physiological significance in management of diabetic retinopathy, rheumatoid arthritis, psoriasis, cardiovascular diseases, and cancer. http://www.jmolecularsignaling.com/content/3/1/7 Sharmila Shankar, Qinghe Chen and Rakesh K Srivastava Journal of Molecular Signaling 2008, 3:7 2008-03-20 doi:10.1186/1750-2187-3-7 Journal of Molecular Signaling 1750-2187 3 7 2008-03-20 Background: The cellular localization of the α1D-adrenergic receptor (α1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the α1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. Results: The adenovirally expressed α1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α1D-AR was also demonstrated with immunocytochemistry using an α1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α1A-α1B- and α1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α1D-AR heterodimerizes with the β2-AR and that desensitization of the β2-AR results in α1D-AR desensitization. In the rat aorta, desensitization of the β2-AR had no effect on contractile responses mediated by the α1D-AR. Conclusion: Our results suggest that the dimerization of the α1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the α1D-AR. http://www.jmolecularsignaling.com/content/3/1/6 Mary L García-Cazarín, Jennifer L Smith, Kyle A Olszewski, Dan F McCune, Linda A Simmerman, Robert W Hadley, Susan D Kraner and Michael T Piascik Journal of Molecular Signaling 2008, 3:6 2008-02-27 doi:10.1186/1750-2187-3-6 Journal of Molecular Signaling 1750-2187 3 6 2008-02-27 Background: CB1 cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated. Results: A CB1 receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB1 receptor. Conclusion: Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB1 receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues. http://www.jmolecularsignaling.com/content/3/1/5 Lea W Padgett, Allyn C Howlett and Joong-Youn Shim Journal of Molecular Signaling 2008, 3:5 2008-02-26 doi:10.1186/1750-2187-3-5 Journal of Molecular Signaling 1750-2187 3 5 2008-02-26 Wiskott Aldrich Syndrome protein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. Here, using the TAT-transduction method, we show the possible role of WASP domain(s) in sealing ring formation and bone resorption. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption. Transduction of WASP peptides containing basic, verpolin-central, pTyr294, and proline-rich regions inhibited the processes listed above at various levels. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated the following: a) a considerable decrease in the interaction and phosphorylation of c-Src with endogenous WASP; b) total loss of sealing ring-like structures; c) formation of actin-rich patches at the peripheral edge that contains filopodia-like projections; d) reduced capacity for bone resorption in vitro. These findings suggest that modulation of phosphorylation state of pTyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring. http://www.jmolecularsignaling.com/content/3/1/4 Tao Ma, Venkatesababa Samanna and Meenakshi A Chellaiah Journal of Molecular Signaling 2008, 3:4 2008-02-20 doi:10.1186/1750-2187-3-4 Journal of Molecular Signaling 1750-2187 3 4 2008-02-20