Top PDF Regulator of G-protein signaling - 5 (RGS5) is a novel repressor of hedgehog signaling.

Regulator of G-protein signaling - 5 (RGS5) is a novel repressor of hedgehog signaling.

Regulator of G-protein signaling - 5 (RGS5) is a novel repressor of hedgehog signaling.

We provide evidence that RGS proteins regulate canonical Hh signaling at the level of Smo-mediated G-protein coupling to downstream effector pathways in mammalian cells. RGS proteins accelerate GTP hydrolysis by Ga proteins and thereby inhibit GPCR-mediated signaling [25]. We found that over-expression of RGS5 inhibits gene expression downstream of Smo in C3H10T1/ 2 cells (Fig. 1B) and functionally inhibits Hh-dependent osteogenic development (Fig. 1D). Conversely, loss of RGS5 function led to increased levels of Shh-stimulated gene expression (Fig. 2B). Moreover, RGS5 was found to be present with acetylated tubulin in the primary cilia (Fig. 3A) and could be Co-IPed in a complex with Smo and acetylated tubulin (Fig. 3B). Taken together, these results demonstrate that RGS5 functions as an inhibitor of Hh signaling downstream of Smo. We propose the interaction between Smo, Ga subunits, and RGS proteins may provide novel targets for the control of Hh-mediated signaling in human disease. Smo is an integral membrane protein with significant structural homology to GPCRs [11–13]. In the unstimulated state, Ptc proteins inhibit Smo signaling, presumably by preventing Smo localization to the primary cilia ([36]; Fig. 4A). However, upon binding Hh proteins (Shh, indian hedgehog (Ihh), or desert hedgehog (Dhh)), Ptc leaves and Smo enters the primary cilia, where it resides in close proximity to other components of the Shh signaling complex: the Gli transcription factors and the large G proteins (Fig. 4B) [1–3,28,70,71]. Multiple recent studies have characterized the interactions between Smo and members of the large G protein family. In Drosophila, Ogden et al demonstrated that Smo signals through Ga i [38]. In mammalian cells, Riobo
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Synergism between Hedgehog-GLI and EGFR signaling in Hedgehog-responsive human medulloblastoma cells induces downregulation of canonical Hedgehog-target genes and stabilized expression of GLI1.

Synergism between Hedgehog-GLI and EGFR signaling in Hedgehog-responsive human medulloblastoma cells induces downregulation of canonical Hedgehog-target genes and stabilized expression of GLI1.

experiments were carried out. As long as the HH-pathway was silent, repressor GLI3 protein was observed as the dominant GLI3 form (Figure 1 A). In Shh-N primed Daoy cells, the GLI3 repressor form disappeared so that GLI3A/GLI3R ratios increased and GLI3A became the dominant GLI3 isoform (Figure 1 A). A quantitative and is well-accepted method to directly assess GLI activation in response to pathway activation is determining the ratio between full-length activator GLI3 (GLI3A) and repressor GLI3 (GLI3R) [31] [32] [33]. Processing of GLI3A Figure 1. Daoy cells respond to SAG and Shh-N by upregulating canonical HH/GLI targets. (A) Incubation with SAG (100 nM) induced the expression of GLI1 protein and inhibited processing of endogenous GLI3 to its repressor form. Co-incubation with Cyclopamine (cyc, 5 mM) inhibited the SAG-induced GLI1 expression and promoted processing of GLI3 to its repressor (GLI3R) form and inhibited GLI2 expression. Beta-actin shown as Western blot loading control. (B) Transcripts for GLI1 and PTCH were upregulated after stimulation with Shh-N for 24 h, induction of HHIP transcripts was seen after 48 h. Enhanced GLI1, HHIP, and PTCH expression levels were still observed after 72 hours. (C) Schematic presentation of canonical Hedgehog signaling. Left panel illustrates silencing of Hedgehog-mediated signaling via a PTCH-mediated block of SMO so that repressor GLI3 prevails and limits the expression rate of Hedgehog target genes. The right panel illustrates the ‘‘Hedgehog-on’’ state, activating GLI proteins now control the expression of Hedgehog-target genes such as GLI1, PTCH, and HHIP. Upregulation of PTCH and HHIP will eventually result in the downregulation of Hedgehog-signaling. Red color indicates a protein with inactivating properties, white color indicates a protein with activating properties, and grey color indicates RNA transcripts.
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SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

An exciting finding from the SILAC assay was the identification of several RAB effectors as novel interaction partners of SDCCAG8, none of which had been previously associated with the cilium. We demonstrated enrichment of RABEP2 and ERC1 at the centrosome, and localization of RABEP2 within the cilium by IF. RABEP2 (previously named Rabaptin-5beta, due to 42% identity to Rababtin-5) was originally identified by yeast two-hybrid screen as an effector of RAB5. The protein was shown to interact with the RAB5 exchange factor Rabex-5 and regulate RAB5 mediated endosomal fusion [30]. It contains 3 coiled-coil domains and a C-terminal RAB- binding domain. This work discovered RABEP2domcritical role in ciliogenesis by using siRNA knockdown. Our data suggest that RABEP2 localization at the centrosome is regulated by SDCCAG8, since knockdown of SDCCAG8 abolished RABEP2 centrosomal localization. We also demonstrated that RABEP2 is a component of the cilium, suggesting that it may be a compo- nent of the intraciliary transport mechanism. Further supporting a role of RABEP2 in the cilium is our demonstration that centrosomally targeted RABEP2 failed to rescue the ciliogenesis defect in SDCCAG8 knockdown cells. However, more work is needed to uncover the precise mecha- nism of RABEP2 recruitment at the centrosome and its function within the cilium.
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Stac3 is a novel regulator of skeletal muscle development in mice.

Stac3 is a novel regulator of skeletal muscle development in mice.

In summary, we have demonstrated that the Stac3 gene is exclusively expressed in skeletal muscle and that it is essential for development of functional skeletal muscle and mice viable at birth. The domain structures of Stac3 protein suggest that it is a signaling protein, and further investigation into the molecular and cellular cause of abnormalities in Stac3 mutant skeletal muscle may reveal novel signaling mechanisms that regulate skeletal muscle de- velopment or function. The centrally located myonuclei and lack of muscle contraction phenotypes of Stac3 mutant mice are hallmarks of centronuclear/myotubular myopathies in humans [31,32]. Therefore, the Stac3 mutant mouse may represent a useful animal model for understanding the pathophysiology of this muscle disease. While this manuscript was being prepared, a study published in JBC indicates that Stac3 is essential for myotube formation in zebrafish [33]. Our study, however, shows that Stac3 gene inactivation does not prevent myotube formation in mice (Fig. 3, Fig. 5). The reason for this difference is not clear, but it may be related to the use of different model systems between the two studies.
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In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

RT-PCR was used to measure the abundance of CG13343 and CG7375 mRNA after RNAi manipulation or in loss-of-function mutant alleles. To test RNAi efficiency, RNA were extracted from wing discs (100 pairs per sample) of third-instar larvae that expressed RNAi transgene under the control of the MS1096-Gal4 driver at 29 uC. For characterization of loss-of-function alleles, RNA were extracted from GFP-negative first-instar larvae (40 larvae per sample) of CG13343 SH2028 /CyO, Kr-gfp or CG7375 LL04684 /TM3, twi-gfp flies. Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Contaminated DNA was digested using RNase-free DNase followed by a phenol/chloroform extraction to remove protein. First strand cDNA was synthesized from 1 m g of each sample using SuperScript III reverse transcriptase (Invitrogen). Semi-quantitative PCR was performed utilizing 20–35 cycles. The linear amplification stage for each primer set was determined by running the same volumes of amplified products on an agarose gel. a -tubulin primers were used for loading control. Primers used are listed as follows: 5 9-GGCGTTGTCAAGCACATCATTC-39 and 5 9-TTTATCACATCCTCCAGCGTGG-39 for CG13343 RNAi; 59-GTACGAATTCATGTCTGTCCACTCACCC-39 and 59- CTGATCTAGATAGACCATCTCCACCTCAT-3 9 to amplify full-length CG13343 cDNA in CG13343 SH2028 mutant; 5 9-G- GAARCCAGTGCTGAACATCAACTC-39 and 59-ACGCAT- CGCCTTCTTTACATTG-3 9 for CG7375 RNAi; 59-AGTC- GAATTCAAATGATTAAACTATTCACG-3 9 and 59-ATGC- TCTAGACACTTGAGCAGACAGCACT-39 to amplify full- length CG7375 cDNA in CG7375 LL04684 mutant; 59-GATCGTC- GATCTGGTTCTGGACAG-3 9 and 59-CCAGTGGACGAAG- GCACGCTT-39 for a-tubulin.
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Hedgehog inhibition promotes a switch from Type II to Type I cell death receptor signaling in cancer cells.

Hedgehog inhibition promotes a switch from Type II to Type I cell death receptor signaling in cancer cells.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a potent death ligand that preferentially induces apoptosis in transformed cells, initiates signaling cascades by ligating two death receptors, DR4 (TNFRSF10A, also referred to as TRAIL receptor 1) and DR5 (TNFRSF10B, also referred to as TRAIL receptor 2/KILLER/TRICK-2) [1]. TRAIL-induced clustering and oligomerization of DR4 and DR5 results in conformational changes of the death domains within their cytoplasmic tails, facilitating recruitment and activation of caspases 8 and 10 within a death inducing signaling complex (DISC) [2,3,4]. If activation of these initiator caspases is sufficiently robust, they directly activate caspase 3, which in turn results in cellular demise by the so-called Type I death receptor pathway of apoptosis [5,6]. However, if the magnitude of caspase 8 and 10 activation is not sufficient to directly activate caspase 3, TRAIL may still induce apoptosis through cleavage of the proapoptotic BH3-only protein Bid to generate truncated Bid (tBID). tBid protein triggers mitochondrial outer membrane permeabilization by promoting oligomerization of the pro-apoptotic Bcl-2 family proteins Bak and/or Bax within this membrane. Mitochondrial outer membrane permeabilization results in egress of pro- apoptotic proteins from the mitochondrial intermembrane space (i.e., cytochrome c, Smac/DIABLO, HtrA2, AIF, and endonu- clease G), which culminates in the mitochondrial pathway of
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Hedgehog Is a Positive Regulator of FGF Signalling during Embryonic Tracheal Cell Migration

Hedgehog Is a Positive Regulator of FGF Signalling during Embryonic Tracheal Cell Migration

ptc is a D. melanogaster segment polarity gene detected throughout embryonic development, larval and pupal stages, with no reported maternal contribution [36,37]. To define the domain of ptc expression in embryonic tissues during tracheal development, we used a ptc-lacZ enhancer trap line (Fig. 5 C, D) and a monoclonal antibody against the first extracellular domain of this receptor (Fig. 5 A, B), which in normal conditions detects Ptc predom- inantly in early endocytic vesicles [38]. According to our observations, ptc-lacZ ßgal expression mimics endogenous Ptc protein expression throughout all tracheal development stages (Fig. 5 A–D and Fig. S2). For this reason, we equally used these two approaches to detect Ptc. At stage 11, Ptc was detected in a stripe at the anterior portion of each tracheal placode (Fig. 5 A, B) [39]. Ptc was found in cells surrounding the migrating tracheal branches (Fig. 5 C and Fig. S2). Later on, Ptc was detected in cells surrounding the migrating GBs and LTps at stages 12 to 15 (Fig. 5 C, D, arrows and Fig. S2). In many cases, tracheal cells extended in very close contact to cells strongly expressing Ptc (Fig. 5 C, D). Using the Ptc antibody, we were unable to detect Ptc expression in Figure 2. ptc mutants have fewer tracheal cells than wild-type. (A–C) Lateral views of Tr4-Tr7 of stage 13 wt (A), ptc mutant (B) and cycA mutant (C) embryos stained with anti-Trh antibody to visualise the tracheal nuclei. Nuclei were marked and counted using the Imaris software and the numbers in each metamere correspond to the number of nuclei marked. Anterior is left. (D–F) Ventral views of stage 16 wt (D), ptc mutant (E) and cycA mutant (F) whole-mount embryos stained with 2A12 antibody to visualise the tracheal lumen. GBs are present and enter the VNC in cycA mutant embryos, despite the very low numbers of tracheal cells. Anterior is left. (G,H) Quantification of tracheal cell numbers in wt, ptc and cycA embryos. (G) Comparison of cell numbers in Tr5 of wt and ptc mutant at stages 11 and 13; Tr5 at stage 11 have an average of 90 cells in the wt (n = 16) and 73 cells in ptc embryos (n = 18); Tr5 at stage 13 have an average of 97 cells in the wt (n = 11) and 63 cells in ptc embryos (n = 19). (H) Comparison of cell numbers in Tr5 of wt, ptc and cycA mutants at stage13; Tr5 at stage 11 have an average of 97 cells in the wt (n = 11), 63 cells in ptc embryos (n = 19) and 38 cells in cycA mutants (n = 8). *P-value#0.01.
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Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension.

Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension.

Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat mod- els of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1)/activator of G protein signaling 8 (Ags8) to be the strongest candidate for C1QTL1, since numerous non- synonymous mutations are found. Moreover, the 5’ Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evi- dence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or tar- geted-gene modifications, it might represent a part of the BP genetic architecture that oper- ates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could vali- date the concept that a QTL in itself can influence BP ‘indirectly’ by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 varia- tions will reveal novel insights into the BP modulation via a regulatory hierarchy.
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Sfrp5 modulates both Wnt and BMP signaling and regulates gastrointestinal organogenesis [corrected] in the zebrafish, Danio rerio.

Sfrp5 modulates both Wnt and BMP signaling and regulates gastrointestinal organogenesis [corrected] in the zebrafish, Danio rerio.

To determine if defects in endodermal organ specification might also result in defects in organ specification, we chose four markers of mature organ function: fatty acid binding protein 10a (fabp10a) for liver, annexin A2b (anxa2b) for intestine, trypsin (try) for exocrine pancreas, and preproinsulin (ins) for endocrine pancreas. We injected 1–2 cell embryos with either a morpholino against sfrp5 or 50 pg of sfrp5 mRNA. Analysis of morpholino injected embryos at 3 dpf by whole mount in situ hybridization showed that the size of liver, pancreas, and intestine was markedly decreased (Fig. 7). Similar to Figure 2. Overexpression of sfrp5 disrupts gastrulation. Embryos were injected with 100 pg mCherry as control (A, E, I, M, Q, U, Y, AA, AC), 140 pg sfrp5 (B, F, J, N, R, V, Z, AB, AD), 150 pg dvl2DDEP (C, G, K, O, S, W), or 50 pg chd (D, H, L, P, T, X). A–D) Morphology of injected embryos injected at early somitogenesis, lateral view with dorsal side to right. E–AD) Whole-mount in situ hybridization of injected embryos. E–H) Animal pole view with dorsal side to the right of embryos stained with eve1 and gsc probes at shield stage. Arrowheads demarcate gsc staining. I–L) Animal pole view with dorsal side to the right of embryos stained with chd, demarcated by arrowheads, at shield stage. M–T) Early somitogenesis embryos stained with probes against ctsl1b, dlx3b, and ntla. M–P: Lateral view with dorsal to top. Arrowheads mark length of notochord. Q–T: dorsal view with anterior to top. Brackets show notochord width. U–X) Mid-somitogenesis embryos stained with egr2b and myoD1. Dorsal view with anterior to top. In X, arrows mark radialization of egfr2b and myoD1 staining. Y, Z) Bud-stage embryos hybridized with probe against her5; anterior view, dorsal to bottom. AA–AB) Early somitogenesis embryos hybridized with probes against ctsl1b, dlx3b, and ntla. Arrow: normal ctsl1b staining. Arrowhead: ectopic ctsl1b staining. AA: Dorsal view, anterior to top. AB: Ventral view, dorsal to top. AC–AD) Mid-somitogenesis embryos hybridized with probes against egr2b and myoD1. AC: Dorsal view, anterior to top. AD: ventral view, dorsal to top. Arrows point to rhombomere 3.
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Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

PhLP1 F/F Cre + mice were bred with Gnat1 -/- mice to create a double knockout PhLP1 F/F Cre + Gnat1 -/- to remove rod signaling that could interfere with cone-driven optomotor responses. Photopic visual acuity and contrast sensitivity of PhLP1 F/F Cre + Gnat1 -/- and PhLP1 +/+ Cre + Gnat1 -/- mice were measured using a two-alternative forced-choice protocol [26]. The Opto- motry system (Cerebral Mechanics) consisted of a square array of four computer monitors with a pedestal in the center where the mouse was placed. A television camera mounted above the animal was used to observe the mouse but not the monitors. Using a staircase paradigm, rotating stimuli (sine-wave vertical gratings) were applied on the monitors where they formed a virtual cylinder around the mouse [27]. The mouse responded to the stimuli by reflexively moving its head in the direction of the rotation. Optomotor responses were measured under photopic background illumination (1.85 log cd m -2 ).
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Vimar Is a Novel Regulator of Mitochondrial Fission through Miro.

Vimar Is a Novel Regulator of Mitochondrial Fission through Miro.

We cannot test effect of GOF vimar under the Miro RNAi background, because Miro RNAi did not induce the wing posture defects in the flight muscles. To further test Miro/vimar inter- action, we generated transgenes of constitutively GDP-bound or GTP-bound mutant of Miro. The rational is that GOF or LOF vimar should not affect these mutant phenotypes if vimar functions as a Miro GEF. Based on a previous report [40], the amino acid substitutions of A20V (Miro20V) and T25N (Miro25N) should render Miro constitutively GTP-bound and GDP-bound, respectively. As expected, a Miro25N overexpression in the flight muscle (Mhc>Miro25N) did not affect the wing posture (Fig 2E) or mitochondria morphology (Fig 2Fa and 2Fb). In contrast, a strong wing posture defect (Fig 2E) and enlarged mitochondria size (Fig 2Fc) were observed in the Miro20V overexpression line (Mhc>Miro20V). Impor- tantly, GOF or LOF vimar failed to affect the defects in the Miro20V overexpression line (Fig 2E, 2Fd and 2Fe). We also examined vimar effect on mitochondrial transport in the GOF Mir- oWT, Miro20V and Miro25N background. However, we found that almost no mitochondria were distributed in the axons in the GOF MiroWT or Miro20V background. This data is con- sistent with previous reports indicating GOF Miro strongly increased mitochondrial length and reduced transportation [41, 42]. We could not examine their mitochondrial transports. In contrast, mitochondrial transport was unaltered under GOF vimar background or combined with Miro25N expression (S2B Fig). Together, these results suggest that vimar requires the nor- mal GTP/GDP binding activity of Miro for its function.
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A novel effect of geraniin on OPGRANKL signaling in osteoblasts

A novel effect of geraniin on OPGRANKL signaling in osteoblasts

RAW264.7 cells are the only osteoclast precursor cells and share an even deeper homology with MC3T3-E1 cells (Sanchez-Fernandez et al., 2008). It is reported that RAW264.7 cells can express the marker gene of osteoclastic phenotype and shows the function of bone resorption (Walsh et al., 2003). In this study, OLCs were successfully induced from RAW264.7 by RANKL and M-CFS. The fusion index (FI), presented as the average number of mOC cultured in vitro, indicates the number of cells that participate in cell fusion and mOC formation. TRAP staining, morphological observation and formation of bone resorption lacunae have been used as the important and reliable method for identifying OC (Walsh et al., 2003). Our results revealed that geraniin significantly decreased the number of mOC and pOC, and the inhibited osteoclast formation due to its suppressing the fusion of pOC into mOC.
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HIV-1 activates macrophages independent of Toll-like receptors.

HIV-1 activates macrophages independent of Toll-like receptors.

were washed twice with phosphate-buffered saline (PBS), pH 7.4, and immediately lysed in ice-cold lysis buffer (1% Triton X-100, 2 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1 mM NaF, 1mM Sodium Orthovanadate, 1 Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland), 1% Phosphatase Inhibitor Cocktail I (Sigma-Aldrich), and 1% Phosphatase Inhibitor Cocktail II (Sigma-Aldrich)). Whole-cell lysates were centrifuged at 6,0006 g for 10 min at 4uC, and the supernatants were frozen at 280uC. The protein concentrations of cell extracts were determined by the BCA protein assay (Bio-Rad, Hercules, CA). Aliquots of cell extracts containing 15 mg of total proteins were diluted in Laemlli Sample Buffer with 5% b- mercaptoeth- anol, and resolved on 4–20% sodium dodecyl sulfate-polyacryl- amide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% milk in TTBS (10 mM Tris, pH 7.4, 100 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature and probed with mouse anti-IRAK-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-pIRF-3 (Cell Signaling Technology, Danvers, MA), mouse anti-IRF-3 (Santa Cruz Biotechnology), goat anti-Mx1 (Santa Cruz Biotechnology), or mouse anti-b-actin (Santa Cruz Biotech- nology), followed by goat anti-mouse IgG-HRP (Cell Signaling Technology), mouse anti-goat IgG-HRP (Santa Cruz Biotechnol- ogy), or goat anti-rabbit IgG-HRP (Cell Signaling Technology). Horseradish peroxidase activity was visualized through enhanced chemiluminescence detection Visualizer (Upstate, Charlottesville, VA) for low abundant proteins, such as p-IRF-3, or Luminol (Santa Cruz Biotechnology) followed by exposure to CL-Xposure film (Thermo Scientific, Waltham, MA). Relative abundance of bands was measured in Adoby Photoshop CS3 (San Jose, CA). Density of individual bands were normalized to respective actin for each lane.
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The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

pectoral fins, which is disrupted in syu, con, and smu [26,60], appears normal in you embryos. you mutants, therefore, show characteristic Hedgehog signaling defects in slow muscle specification, patterning of ventral spinal cord, and the development of the dorsal aorta, but you is apparently not required for Hedgehog signaling in some other regions of the zebrafish embryo. Because the primary cell types disrupted in you mutants all develop in close proximity to the notochord, it is possible that you gene function may be required for the transport or stability of Hedgehog signals in the vicinity of the developing notochord but not some other regions. The notochord is a defining feature of chordates, and a notochord-associated function would explain why no you counterpart is required for Hedgehog signaling in the fly. It is not clear, however, why Hedgehog signaling near the notochord would require a special extracellular mediator. Another possibility is that maternal you function could mask earlier requirements in zygotic you mutants; future work with maternal-zygotic you mutants is needed to address this possibility. A third explanation of the requirement for you in only a subset of Hedgehog-regulated processes is that additional factors with redundant functions may substitute for you in other regions of the embryo. Intriguingly, expression of another Scube gene in mouse—Scube1—is observed in many embryonic tissues known to require Hedgehog signaling for their proper development, including the ventral forebrain, limb bud, somites, and developing gonad [48]. These results suggest that an additional zebrafish Scube gene may also play a role in the development of other areas of the embryo where Hedgehog signaling is active. Moreover, interactions between SCUBE proteins may be important for Hedgehog signaling; biochemical analysis suggests that SCUBE1 and SCUBE2 proteins can interact to form both homodimers and heterodimers [52].
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Discovery of novel small molecule activators of β-catenin signaling.

Discovery of novel small molecule activators of β-catenin signaling.

Despite the absence of b-catenin activation in any cell line other than U2OS-EFC cells and U2OS-Da-Nuc cells, our data do not suggest that activation of b-galactosidase activity by Cpd1 and Cpd2 is an assay artifact. Firstly, other parameters of Wnt/b- catenin signaling, such as b-catenin-dependent reporter genes and Western blotting of cytoplasmic b-catenin levels also imply activation of Wnt/b-catenin signaling in these cells (Figure 1C– E, Figure 4A). As an additional control experiment, we transfected U2OS-Da-Nuc cells with luciferase reporter genes sensitive to cAMP (CREB), Ca 2+ (NFAT) and glucocorticoid hormone receptor (GR) signaling. None of these reporter genes was activated by Cpd1, whereas SuperTOPflash reporter gene activity was dose-dependently induced by Cpd1 treatment (Figure S5A– D). Furthermore, HEK293 cells engineered to complement b- galactosidase in response to rmWnt-3a (HEK293-EFC cells) did not activate b-catenin signaling when treated with Cpd1 or Cpd2 (Figure 4D). In addition, Cpd1 did not induce b-galactosidase activity in a cellular EFC assay for the recruitment of the scaffolding protein b-arrestin2 to human parathyroid hormone receptor 1 (Figure S6). Furthermore, Cpd1 did not induce the nuclear translocation of human GR in U2OS cells (U2OS-GR)
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J. Appl. Oral Sci.  vol.20 número2

J. Appl. Oral Sci. vol.20 número2

This suggests that modulation of some cell signaling pathways may have negative effects; however another hallmark of cytokine cell signaling is that activation of these pathways by c[r]

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Identification of proteins with potential osteogenic activity present in the water-soluble matrix proteins from crassostrea gigas nacre using a proteomic approach

Identification of proteins with potential osteogenic activity present in the water-soluble matrix proteins from crassostrea gigas nacre using a proteomic approach

Altogether the available information on canonical Wnt signaling as a bone formation regulator and the identification in nacre WSM (Figure 1) of proteins homologous to Wnt inhibitory factor-1 leads us to suggest that Wnt antagonists present in nacre may be related to the WSM nacre known osteogenic activity although further studies are necessary in order to understand the role of WIF-1 in bone development. Tenascins are a glycoprotein family associated with the organic extracellular matrix (ECM), that induce prolifera- tion, di fferentiation and cellular migration [ 41–43]. Four members of this family (tenascins C, R, X and W) have been identified and characterized in vertebrates [44]. The basic structure of tenascins are composed by epidermal growth factor (EGF) repeats in the direction of the amino end, fibronectin type III domains and a globular fibrinogen domain in the carboxylic end. Differences in both the num- ber and nature of EGF and fibronectin type III domains can be observed between different species [41, 42]. Tenascin C is a disulfide hexamer, with a cysteine-rich center, composed by subunits with a molecular mass of approximately of 200 kDa. These subunits can have different masses due to glycosylation [41]. In mammals, the subunits are constituted by 14.5 EGF repeats with 8 fibronectin type III domains present in all the tenascin C isoforms [45].
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Retroactive signaling in short signaling pathways.

Retroactive signaling in short signaling pathways.

In Ossareh et al, the authors performed mathematical analysis of retroactivity in a signaling cascade with an arbitrary number of stages. They achieved necessary and sufficient conditions for which retroactivity exists in such chains. Their analysis is based on the linearization of the steady state equations in order to predict how a small downstream perturbation is amplified in the upstream response of an arbitrarily long signaling chain. Those results are complementary to the ones presented in the present paper, in the sense that here we consider short signaling pathways but our analysis is based on the resolution of the full nonlinear equations, and not only on the linearized system. So, it is concerned with arbitrarily large perturbations of the parameters. In fact we show that retroactive signaling is meant to work only for a characteristic range of parameter variations that we analytically estimate by working on the asymptotic behaviors of the system for small and large parameter perturbations.
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The adaptor protein myd88 is a key signaling molecule in the pathogenesis of irinotecaninduced intestinal mucositis

The adaptor protein myd88 is a key signaling molecule in the pathogenesis of irinotecaninduced intestinal mucositis

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARRIVE Guidelines (Animals in Research: Reporting In Vivo Experiments) [23]. All efforts were made in order to minimize animal suffering. In the survival study, the animals were monitored twice daily for ten days following the first injection of irinotecan. During the experiment, fifty per- cent of the animals succumb due to the treatment and its consequences, including diarrhea. Those animals that showed signs of imminent death, including piloerection, reduced locomo- tion, inability to maintain upright position, ataxia, tremor and altered breath frequency were euthanized by ketamine/xylazine overdose (>100/10 mg/kg, s.c., União Química, São Paulo, Brazil) followed by cervical dislocation. Pain relievers or anesthesia were not used in our exper- iments since those agents directly interfere with the production of inflammatory mediators and/or alter the gastrointestinal transit and mask the diarrheic events in this animal model. At the end of the survival experiment, live animals were euthanized by ketamine/xylazine over- dose (>100/10 mg/kg, s.c., União Química, São Paulo, Brazil) followed by cervical dislocation. The experimental protocol, including the mortality aspects of the protocol, was reviewed and approved by the Committee on the Ethics of Animal Experiments of the Federal University of Ceará (Permit Number: 99/10).
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The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.

The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.

Here, we demonstrate that in contrast to the V protein of MuV, TioV-V hardly interacts with human STAT2, does not induce STAT1 degradation and is unable to block signal transduction downstream of IFN-a/b and IFN-l receptors. However, TioV-V remains functional regarding other known activities of rubulavirus V proteins, and besides interactions with MDA5 and LGP2, it was found to bind DDB1 and STAT3. This later interaction probably Figure 7. TioV-V fails to interact with human STAT2 and does not induce STAT1 degradation. (A–B) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V, TioV-V (A–B) or NiV-V (B) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human STAT2 (A) or STAT1 (B). Total cell lysates from transfected cells were prepared at 48 h post- transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull- down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. (C) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V, MuV-V or CHIKV-nsP4 (500 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG- tagged human STAT1 and STAT2 (150 ng/well of each vector). At 24 h post-transfection, cells were left untreated or stimulated with recombinant IFN-b at 200 IU/ml. Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panels). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. Upper and lower panels on top of figure C correspond to short and longer exposures of the same blot, respectively. (D) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 mg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V, TioV-P or TioV-W. Total cell lysates were prepared at 48 h post-transfection and endogenous STAT1 expression levels were determined by western- blot analysis. Actin expression was determined and used as a protein extraction and loading control. (E) HEK-293T cells were transfected with pCI- neo-3xFLAG expression vector (1 mg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V or CHIKV-nsP4. Total cell lysates were prepared at 48 h post-transfection, and 3xFLAG-tagged viral proteins were purified using anti-FLAG antibodies conjugated to sepharose beads. Co- immunopurification of endogenous STAT2 with 3xFLAG-tagged viral proteins was determined by western-blot analysis (top and middle panel, respectively). Actin expression was determined prior to the immunoprecipitation on total cell lysates and used as a protein extraction control (lower panel).
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