Search

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix-cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and mitogen-activated protein kinase activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of pattern-triggered immunity. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitor compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses. Further investigation is needed to understand how cutin breakdowns are perceived and to explore their potential use in agriculture.

Abstract Background Oocytes of large animal species isolated from small ovarian follicles (

Fresh horticultural products are economically significant foods that are highly demanded by consumers worldwide; however, they are highly perishable and susceptible to deterioration by fungi, which contribute to their short shelf-life and cause significant post-harvest losses. Among the alternatives suggested for fungal control in plants is the elicitation of the innate plant defense mechanism, which may be activated when specific molecules of the phytopathogen, such as chitin, are recognized. Chitin is a long-chain polymer of N-acetyl-α-D-glucosamine of the fungal cell wall; it possesses biological activity by eliciting the plant immune response. This molecule and its oligosaccharides are recognized through transmembrane receptors known as receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Mediated by receptor-like cytoplasmic kinases (RLCKs), which bind to the intracellular domain of these receptors, they initiate intracellular signal transduction via MAP kinases, triggering the plant defense response. In model plants, such as Oryza sativa (rice) and Arabidopsis thaliana, the set of RLK/RLP-RLCK-MAP kinases is involved in plant immunity triggered by chitin. Furthermore, in horticultural products, research into the molecular events between these three elements has suggested that similar processes occur. However, little is known about these molecular events in fruits. Against this background, the present review provides the most recent and relevant findings on the molecular associations of these three elements in the response to fungal chitin in plants and outlines which elements could participate in this signaling process in horticultural crops. [ABSTRACT FROM AUTHOR]

Fresh horticultural products are economically significant foods that are highly demanded by consumers worldwide; however, they are highly perishable and susceptible to deterioration by fungi, which contribute to their short shelf-life and cause significant post-harvest losses. Among the alternatives suggested for fungal control in plants is the elicitation of the innate plant defense mechanism, which may be activated when specific molecules of the phytopathogen, such as chitin, are recognized. Chitin is a long-chain polymer of N-acetyl-α-D-glucosamine of the fungal cell wall; it possesses biological activity by eliciting the plant immune response. This molecule and its oligosaccharides are recognized through transmembrane receptors known as receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Mediated by receptor-like cytoplasmic kinases (RLCKs), which bind to the intracellular domain of these receptors, they initiate intracellular signal transduction via MAP kinases, triggering the plant defense response. In model plants, such as Oryza sativa (rice) and Arabidopsis thaliana, the set of RLK/RLP-RLCK-MAP kinases is involved in plant immunity triggered by chitin. Furthermore, in horticultural products, research into the molecular events between these three elements has suggested that similar processes occur. However, little is known about these molecular events in fruits. Against this background, the present review provides the most recent and relevant findings on the molecular associations of these three elements in the response to fungal chitin in plants and outlines which elements could participate in this signaling process in horticultural crops.

We recently reported an RAF rearrangement without NRAS or BRAF mutations in lesions from Giant Congenital Melanocytic Nevi (CMN). The new gene fusion involves the 5′‐end of the promoter‐containing N terminus of the SOX5 gene fused to exons 7–16 of the 3′‐end of RAF1 gene leading to a SOX5‐RAF1 fusion transcript which loses the auto‐inhibitory CR1 domain but retains the complete in‐frame coding sequence for the C‐Terminal kinase domain of the RAF1. Stable expression of SOX5‐RAF1 fusion induced growth factor‐independent cell growth in murine hematopoietic Ba/F3 cells and melan‐a immortalized melanocytes. Besides, it led to the transformation of both Ba/F3 and NIH 3T3 cells as revealed by colony formation assays. Furthermore, its expression results in MAPK activation assessed by increased levels of p‐ERK protein in the cytosol of transduced cells. Treatment with Sorafenib and UO126 inhibited proliferation of Ba/F3‐SOX5‐RAF1 cells in the absence of IL3 but not the PLX 4720, a specific inhibitor of BRAF. Moreover, the tumorigenic and metastatic capacities of SOX5‐RAF1 were assessed in vivo. These results indicate that SOX5‐RAF1, a driver event for CMN development, has oncogenic capacity. Thus, sequencing of CMN transcriptomes may lead to the identification of this druggable fusion and interfere with the progression toward melanoma. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the underlying mechanism of multi-walled carbon nanotubes (MWCNT) induced cellular response and their potential cross-talk, specifically, between endoplasmic reticulum (ER) stress, MAPK activation and apoptosis and how these nano-bio interactions depend on the physico-chemical properties of MWCNT. For this purpose, human bronchial epithelial (Beas2B) and human hepatoma (HepG2) cell lines, were exposed to five kinds of MWCNTs which differ in functionalization and aspect ratios. Tissue-specific sensitivity was evident for calcium homeostasis, ER-stress response, MAPK activation and apoptosis, which further depended on surface functionalization as well as aspect ratios of MWCNT. By applying specific pharmaceutical inhibitors, relevant biomarkers gene and proteins expressions, we found that possibly MWCNT induce activation of IRE1a-XPB1 pathway- mediated ER-stress response, which in turn trigger apoptosis through JNK activation in both type of cells but with variable intensity. The information presented here would have relevance in better understanding of MWCNT toxicity and their safer applications. [ABSTRACT FROM AUTHOR]

Monica Madalina Tucureanu,1,* Daniela Rebleanu,1,* Cristina Ana Constantinescu,1,2 Mariana Deleanu,3,4 Geanina Voicu,1 Elena Butoi,1 Manuela Calin,1 Ileana Manduteanu1 1Department of Biopathology and Therapy of Inflammation, Nicolae Simionescu Institute of Cellular Biology and Pathology, Bucharest, Romania; 2Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania; 3Department of Lipidomics, Nicolae Simionescu Institute of Cellular Biology and Pathology, Bucharest, Romania; 4Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania *These authors contributed equally to this work Background: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of Gi protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin. Purpose: The aim of the present work was to evaluate the potential of a Gi-protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages. Materials and methods: Guanosine 5´-O-(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1β, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated. Results: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1β, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS. Conclusion: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages. Keywords: guanosine 5´-O-(2-thiodiphosphate) (GOT), MAPK activation, cytokine, monocyte adhesion, chemotaxis

Pseudomonas aeruginosa is an opportunistic pathogen and is the major cause of corneal infection worldwide that secret several virulent toxins through its type III secretion system (T3SS). In defense against pathogenic insults, epithelial cells and macrophages express antimicrobial peptides (AMPs) that are essential components of host immune response. In this study, we have determined the expression of several AMPs in patients with P. aeruginosa corneal infection. We also used an in vitro model of infection using human corneal epithelial cells and macrophages to determine the gene expression of AMPs and cellular response to wild-type and T3SS mutant P. aeruginosa. We found differential expression of several AMPs in patient samples and also found that P. aeruginosa repress AMP expression in both epithelial cells and macrophages by its T3SS in vitro. It dampens AMP expression by causing delay in NF-κB, p38 and ERK activation and inhibits reactive oxygen species generation in these cells by its T3SS. Our study show the profile of AMPs expressed during P. aeruginosa keratitis and suggest the pivotal role of the T3SS in epithelial cells and macrophages during P. aeruginosa infection. [ABSTRACT FROM AUTHOR]

The nature of toxic tau in Alzheimer’s disease has been unclear. During pathogenesis, the importance of tau oligomerization versus tau phosphorylation is controversial and the investigation of both remains critical towards defining the toxicity of tau. The phosphorylation of tau on serines and/or threonines occurs early in the disease course and altering phosphorylation has been shown to disrupt neuropathogenesis. We have recently reported that in PC12-derived cells, tau had a role in signal transduction processes activated by NGF. By depleting tau, NGF-induced MAPK activation was attenuated and by restoring tau, MAPK activation was restored. Furthermore, the phosphorylation of tau on thr231 was required for tau to potentiate MAPK activation. Here we report the effects of additional disease-related tau phosphorylation sites and tau isoform on the ability of tau to potentiate MAPK activation. Our findings, which tested three other sites of phosphorylation, showed that phosphorylation at these other sites mainly lessened MAPK activation; none potentiated MAPK activation. In comparing 0N3R tau to the other five brain tau isoforms, most showed a trend towards less MAPK activation, with only 2N4R tau showing significantly less activation. Since MAPK activation has been reported in Alzheimer’s disease brain and is characteristic of cell proliferation mechanisms, tau phosphorylation that promotes MAPK activation could promote cell cycle activation mechanisms. In neurons, the activation of the cell cycle leads to cell death, suggesting that abnormally phosphorylated tau can be a toxic species. The relationship between tau oligomerization and its ability to potentiate MAPK activation needs to be determined.

Vertebrate β-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four β-catenin-related proteins present in the round worm Caenorhabditis elegans. The fourth β-catenin-related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription ofWnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein.We showed previously thatWRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate β-catenin. [ABSTRACT FROM AUTHOR]

Abstract: The dual PI3K–mTOR inhibitor BEZ235 was evaluated in preclinical models of nasopharyngeal carcinoma (NPC). The IC50 value of BEZ235 for growth was in the nanomolar range in vitro, induce G1 cycle arrest and apoptosis, and inhibited AKT and mTOR signaling in most NPC cell lines. No synergistic effect was observed when BEZ235 was combined with chemotherapy. BEZ235 increased MAPK activation in vitro but not in vivo. A daily schedule was more effective than a weekly schedule on tumor growth and inhibition of downstream mTOR signaling in vivo. The activity of BEZ235 maybe independent of the PIK3CA amplification and mutation status. [Copyright &y& Elsevier]

Receptor activator of NF-κ?B ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca2+ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca2+ /calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca2+ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca2+ mobilization induced by aluminum fluoride (AlF4 -), a G-protein activator, with or without RANKL and the effects of AlF4 - on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4 - induces intracellular Ca2+ concentration ([Ca2+]i) oscillations, which is dependent on extracellular Ca2+ influx. Notably, co-stimulation of AlF4 - with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4 -. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca2+]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis. [ABSTRACT FROM AUTHOR]

The nature of "toxic" tau in Alzheimer's disease (AD) has been unclear. During pathogenesis, the importance of tau oligomerization vs. tau phosphorylation is controversial and the investigation of both remains critical toward defining the "toxicity" of tau. The phosphorylation of tau on serines and/or threonines occurs early in the disease course and altering phosphorylation has been shown to disrupt neuropathogenesis.We have recently reported that in PC12-derived cells, tau had a role in signal transduction processes activated by NGF. By depleting tau, NGF-induced MAPK activation was attenuated and by restoring tau, MAPK activation was restored. Furthermore, the phosphorylation of tau on Thr231 was required for tau to potentiate MAPK activation. Here we report the effects of additional disease-related tau phosphorylation sites and tau isoform on the ability of tau to potentiate MAPK activation. Our findings, which tested three other sites of phosphorylation, showed that phosphorylation at these other sites mainly lessened MAPK activation; none potentiated MAPK activation. In comparing 0N3R tau to the other five brain tau isoforms, most showed a trend toward less MAPK activation, with only 2N4R tau showing significantly less activation. Since MAPK activation has been reported in AD brain and is characteristic of cell proliferation mechanisms, tau phosphorylation that promotes MAPK activation could promote cell cycle activation mechanisms. In neurons, the activation of the cell cycle leads to cell death, suggesting that abnormally phosphorylated tau can be a toxic species. The relationship between tau oligomerization and its ability to potentiate MAPK activation needs to be determined. [ABSTRACT FROM AUTHOR]

Abstract: There is increasing evidence that angiotensin II (Ang II), through binding to the type 2 (AT2) receptor may have beneficial effects in various physiological and pathological situations. However, specific action presumably mediated by the angiotensin AT2 receptor has been hampered by the absence of appropriate selective ligands. The aim of this study was to compare the biological properties of two related and selective drug-like nonpeptide AT2 ligands, namely an agonist called M024 (also known as Compound 21) and a new ligand, presumably an antagonist, C38/M132, (originally called C38). Properties of the compounds were investigated in NG108-15 cells expressing angiotensin AT2 receptor and known to develop neurite outgrowth upon Ang II stimulation. NG108-15 cells stimulated for three days with C21/M024 (0.1 or 100nM) exhibited the same neurite outgrowth as cells stimulated with Ang II (100nM) while co-incubation of Ang II or C21/M024 with C38/M132 (10 or 100nM) inhibited their effects, similarly to the angiotensin AT2 receptor antagonist, PD123319 (10μM). As Ang II, C21/M024 induced a Rap1-dependent activation of p42/p44mapk whereas preincubation of cells with C38/M132 inhibited p42/p44mapk and Rap1 activation induced by Ang II. Three-day treatment with C21/M024 or Ang II decreased cell number in culture, an effect that was rescued by preincubation with C38/M132. Taken together, these results indicate that the nonpeptide ligand C21/M024 is a potent angiotensin AT2 receptor agonist while C38/M132 acts as an antagonist. These selective nonpeptide angiotensin AT2 ligands may represent unique and long-awaited tools for the pursuit of in vivo studies. [Copyright &y& Elsevier]

Estrogen-like effects of cadmium (Cd) have been reported in several animal studies, and recent epidemiological findings suggest increased risk of hormone-dependent cancers after Cd exposure. The mechanisms underlying these effects are still under investigation. Our aim was to study the effects of Cd on cellular signaling pathways in vivo with special focus on estrogen signaling and to perform benchmark dose analysis on the effects. Transgenic adult ERE-luciferase male mice were exposed subcutaneously to 0.5–500 μg CdCl2 per kg body weight (bw) or 17α-ethinylestradiol (EE2) for 3 days. These doses had no effects on organ and bw or testicular histology, indicating subtoxic exposure levels. The transgene luciferase, reporting genomic estrogen response, was significantly increased by EE2 but not by Cd. However, Cd significantly affected kinase phosphorylation and endogenous gene expression. Interestingly, gene expression changes displayed a traditional dose-response relationship, with benchmark dose levels for the expression of Mt1, Mt2, p53, c-fos, and Mdm2 being 92.9, 19.9, 7.6, 259, and 25.9 μg/kg bw, respectively, but changes in kinase phosphorylation were only detected at low exposure levels. Phosphorylation of Erk1/2 was significantly increased even in the lowest dose group, 0.5 μg/kg bw, rendering pErk1/2 a more sensitive sensor of exposure than changes in gene expression. Collectively, our data suggest that the effects triggered by Cd in vivo are markedly concentration dependent. Furthermore, we conclude that the estrogen-like effects of Cd are likely to result from a mechanism different from steroidal estrogens. [ABSTRACT FROM PUBLISHER]

Abstract: 6,4′-Dihydroxy-7-methoxyflavanone (DMF), a biologically active compound, was isolated from the heartwood of Dalbergia odorifera T. Chen (Leguminosae). The present study proposed to examine the role of DMF as an anti-oxidative and anti-inflammatory heme oxygenase-1 (HO-1) inducer in mouse hippocampal HT22 cells and BV2 microglia cells. The effect of DMF on cell viability was determined by MTT assay and the effects of DMF on pro-inflammatory enzymes and cytokines were analyzed by western blot and ELISA. Parameters such as DMF induced HO-1 protein immunocontents, HO activity and mitogen-activated protein kinases (MAPK) activation were also measured. DMF increased cellular resistance to oxidative injury caused by glutamate-induced cytotoxicity in HT22 cells, via JUN N-terminal kinase (JNK) pathway dependent HO-1 expression. Furthermore, DMF suppressed the lipopolysaccharide (LPS)-induced expression of pro-inflammatory enzymes and inflammatory mediators in BV2 microglia. DMF suppressed production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), through extracellular signal-regulated kinase (ERK) pathway dependent HO-1 expression. This study indicates that DMF effectively modulates the regulation of anti-oxidative and anti-inflammatory action, via up-regulation of HO-1 in HT22 cells and BV2 microglia. These results suggest that DMF possesses therapeutic potentials against neurodegenerative diseases that are induced by oxidative stress and neuroinflammation. [Copyright &y& Elsevier]

Abstract: The behavioral effects evoked by cannabinoids are primarily mediated by the CB1 and CB2 cannabinoid receptor subtypes. In vitro pharmacology of cannabinoid receptors has been elucidated using recombinant expression systems expressing either CB1 or CB2 receptors, with limited characterization in native cell lines endogenously expressing both CB1 and CB2 receptors. In the current study, we report the molecular and pharmacological characterization of the F-11 cell line, a hybridoma of rat dorsal root ganglion neurons and mouse neuroblastoma (N18TG2) cells, reported to endogenously express both cannabinoid receptors. The present study revealed that both receptors are of mouse origin in F-11 cells, and describes the relative gene expression levels between the two receptors. Pharmacological characterization of the F-11 cell line using cannabinoid agonists and antagonists indicated that the functional responses to these cannabinoid ligands are mainly mediated by CB1 receptors. The non-selective cannabinoid ligands CP 55,940 and WIN 55212-2 are potent agonists and their efficacies in adenylate cyclase and MAPK assays are inhibited by the CB1 selective antagonist SR141716A (SR1), but not by the CB2 selective antagonist SR144528 (SR2). The endocannabinoid ligand 2AG, although not active in adenylate cyclase assays, was a potent activator of MAPK signaling in F-11 cells. The analysis of CB1 and CB2 receptor gene expression and the characterization of cannabinoid receptor pharmacology in the F-11 cell line demonstrate that it can be used as a tool for interrogating the endogenous signal transduction of cannabinoid receptor subtypes. [Copyright &y& Elsevier]

Abstract: Objective. : Epithelial ovarian cancer has the highest mortality rate among gynecologic cancers. Chemotherapy is an essential component of its treatment. While isothiocyanates are known to possess chemopreventive effects against various cancers, yet little is known about their chemotherapeutic potential in ovarian cancer (OC). In the present study, we examined the antiproliferative and apoptotic effect of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate on OVCAR-3 cells. Methods. : Cytotoxic activity of PEITC on OVCAR-3 cells was determined using cell proliferation, apoptosis (DNA fragmentation and TUNEL assay) and caspase-activation studies. The role of PARP-1, Bax, and Bcl-2 in apoptosis was analyzed by Western blotting. Activation of JNK1/2, p38, Akt, ERK1/2, and c-Myc was examined by immunoblotting. Specific inhibitors of caspases, JNK1/2, p38, and MEK were used to corroborate these data. Results. : PEITC was cytotoxic to OVCAR-3 cells, and inhibited proliferation in a dose-dependent fashion (IC50 = 23.2 μM). PEITC induced apoptosis by activating caspase-3 and -9, without capsase-8 activation. Anti-apoptotic Bcl-2 levels were suppressed while pro-apoptotic Bax levels were enhanced. PEITC suppressed activation of Akt, ERK1/2, and the expression of transcription factor c-Myc, while simultaneously activating pro-apoptotic p38 and JNK1/2. Specific inhibitors of caspase-3 and -9, JNK1/2, and p38 reversed the cytotoxic effect of PEITC. Conclusions. : These findings demonstrate that PEITC exhibits cytotoxicity towards OVCAR-3 cells and induces apoptosis via caspase-9 and -3 pathways. PEITC inhibits Akt, ERK1/2 survival signaling, and c-Myc while simultaneously activating pro-apoptotic p38 and JNK1/2. Systematic preclinical and clinical trials with PEITC in ovarian cancer are indicated. [Copyright &y& Elsevier]

Advanced ovarian cancer (OC) is not curable by surgery alone and chemotherapy is essential for its treatment. Isothiocyanates have been shown to inhibit carcinogen-induced tumorigenesis in animal models, yet no efforts have been made to determine their therapeutic potential in OC. In the present study, we investigated the mechanism of the antiproliferative and apoptotic activity of benzyl isothiocyanate (BITC) in OC. BITC inhibited the proliferation of OC cells and induced apoptosis in OC cells. Apoptosis was induced by a strong activation of caspase-3 and -9, and cleavage of PARP-1. However, caspase-8 was not activated by BITC. Cytotoxic effects of BITC were reversed by the inhibition caspase-3 and -9 specific inhibitors. BITC showed a concentration dependent decrease in the levels of Bcl-2 with a concomitant increase in Bax levels. In addition, BITC activated proapoptotic signaling by phosphorylation JNK1/2 and p38 while simultaneously inhibiting survival signaling mediated by ERK1/2 and Akt phosphorylation in a dose-dependent manner. While JNK inhibitor SP600125 and p38 inhibitor SB203580, abolished the cytotoxic effect of BITC, MEK inhibitor, PD98059 and PI3 kinase inhibitor, LY294002 failed to show such reversal indicating a critical role played by JNK1/2 and p38 signaling in apoptosis induced by BITC. In summary, our studies demonstrate that BITC inhibits proliferation of OC cells and induces apoptosis via caspase-9 and -3 pathways. BITC inhibits ERK1/2 and Akt survival signaling while simultaneously activating pro-apoptotic p38 and JNK1/2. Therefore, BITC can be potentially developed as a therapeutic agent to treat OC. [ABSTRACT FROM AUTHOR]

Abstract Recent evidence suggest that many G protein-coupled receptors (GPCR) and signalling molecules localize in microdomains of the plasma membrane. In this study, flotation gradient analysis in the absence of detergents demonstrated the presence of the metabotropic glutamate receptor type 1α (mGlu1α) in low-density caveolin-enriched membrane fractions (CEMF) in permanently transfected BHK cells. BHK-1α cells exhibit a similar pattern of staining for caveolin-1 and caveolin-2, and these two proteins show a high degree of co-localization with mGlu1α receptor as demonstrated by immunogold and confocal laser microscopy. The presence of mGlu1α in CEMF was also demonstrated by co-immunoprecipitation of mGlu1α receptor using antibodies against caveolin proteins. Activation of the mGlu1α receptor by agonist increased extracellular signal-regulated kinases phosphorylation in CEMF and not in high-density membrane fractions (HDMF), suggesting that mGlu1α receptor-mediated signal transduction could occur in caveolae-like domains. Overall, these results clearly show a molecular and functional association of mGlu1α receptor with caveolins. [ABSTRACT FROM AUTHOR]

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11 , plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted. • Disease-associated mutations endow SHP2 liquid-liquid phase separation capability • SHP2 LLPS is driven by electrostatic interactions mediated by PTP domain • SHP2 allosteric inhibitors block SHP2 LLPS by locking SHP2 in closed conformation • Mutant SHP2 can recruit and activate WT SHP2 in LLPS to promote MAPK activation Disease-associated mutants of a critical phosphatase in the RAS-MAPK pathway undergo phase separation through a dominant gain-of-function mechanism, explaining how both enzymatically activating and inactivating mutations dysregulate the pathway and can be therapeutically targeted. [ABSTRACT FROM AUTHOR]

Minocycline, an FDA-approved second-generation semisynthetic tetracycline, exerts antioxidant, anti-apoptotic and anti-inflammatory effects, independent of its antimicrobial properties. Interleukin (IL)-17A is an immune and inflammatory mediator, and its sustained induction is associated with various cardiovascular diseases. Here we investigated (i) whether IL-17A induces cardiomyocyte contractile depression and death, (ii) whether minocycline reverses IL-17A's negative inotropic effects and (iii) investigated the underlying molecular mechanisms. Indeed, treatment with recombinant mouse IL-17A impaired adult cardiomyocyte contractility as evidenced by a 34% inhibition in maximal velocity of shortening and relengthening after 4 h (P <.01). Contractile depression followed iNOS induction at 2 h (2.13-fold, P <.01) and NO generation at 3 h (3.71-fold, P <.01). Further mechanistic investigations revealed that IL-17A-dependent induction of iNOS occurred via TRAF3IP2, TRAF6, TAK1, NF-κB, and p38MAPK signaling. 1400 W, a highly specific iNOS inhibitor, suppressed IL-17A-induced NO generation and contractile depression, where as the NO donors SNAP and PAPA-NONOate both suppressed cardiomyocyte contractility. IL-17A also stimulated cardiomyocyte IL-1β and TNF-α secretion, however, their neutralization failed to modulate IL-17A-mediated contractile depression or viability. Further increases of IL-17A concentration and the duration of exposure enhanced IL-1β and TNF-α secreted levels, buthad no impact on adult cardiomyocyte viability. However, when combined with pathophysiological concentrations of IL-1β or TNF-α, IL-17A promoted adult cardiomyocyte death. Importantly, minocycline blunted IL-17A-mediated deleterious effects, indicating its therapeutic potential in inflammatory cardiac diseases. Unlabelled Image • Interleukin-17A impairs adult cardiomyocyte contractility, but not death. • IL-17A induces cardiomyocyte death when combined with pathophysiological levels of IL-1β and TNF-α. • IL-17 induces cardiomyocyte contractile depression via iNOS induction and NO generation. • Minocycline inhibits IL-17A-induced iNOS expression and NO generation. • Minocycline inhibits IL-17A-induced cardiomyocyte contractile depression and death. [ABSTRACT FROM AUTHOR]

Rationale: Glioblastoma multiforme (GBM) almost invariably gain invasive phenotype with limited therapeutic strategy and ill-defined mechanism. By studying the aberrant expression landscape of gliomas, we find significant up-regulation of p -MAPK level in GBM and a potent independent prognostic marker for overall survival. DHHC family was generally expressed in glioma and closely related to the activation of MAPK signaling pathway, but its role and clinical significance in GBM development and malignant progression are yet to be determined. Method: Bioinformatics analysis, western blotting and immunohistochemistry (IHC) were performed to detect the expression of ZDHHC17 in GBM. The biological function of ZDHHC17 was demonstrated by a series of in vitro and in vivo experiments. Pharmacological treatment, flow cytometry, Transwell migration assay, Co- Immunoprecipitation and GST pulldown were carried out to demonstrate the potential mechanisms of ZDHHC17. Results: ZDHHC17 is up-regulated and coordinated with MAPK activation in GBM. Mechanistically, ZDHHC17 interacts with MAP2K4 and p38/JNK to build a signaling module for MAPK activation and malignant progression. Notably, the ZDHHC17-MAP2K4-JNK/p38 signaling module contributes to GBM development and malignant progression by promoting GBM cell tumorigenicity and glioma stem cell (GSC) self-renewal. Moreover, we identify a small molecule, genistein, as a specific inhibitor to disrupt ZDHHC17-MAP2K4 complex formation for GBM cell proliferation and GSC self-renewal. Moreover, genistein, identified herein as a lead candidate for ZDHHC17-MAP2K4 inhibition, demonstrated potential therapeutic effect in patients with ZDHHC17-expressing GBM. Conclusions: Our study identified disruption of a previously unrecognized signaling module as a target strategy for GBM treatment, and provided direct evidence of the efficacy of its inhibition in glioma using a specific inhibitor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

A key member of the sentrin/small ubiquitin-like modifier (SUMO)-specific protease (SENP) family, SENP2 has been shown to implicate embryonic development, fatty acid metabolism, atherosclerosis, and neurodegenerative diseases. However, other biologic functions of SENP2 and its specific targets are incompletely understood. Here, we uncovered a novel role of SENP2 in negative regulation of keratinocyte migration, a process crucial to wound epithelialization. Defects in this function are often associated with the clinical phenotypes of chronic nonhealing wounds. Mechanistically, SENP2 as a specific de-SUMOylase targets NDR1 (nuclear Dbf2-related 1), also called STK38 (serine-threonine kinase 38), for de-SUMOylation and SUMO conjugation of NDR1 on Lys-465 attenuates its inhibition of p38/ERK1/2 activation by decreasing the association of NDR1 with MEK kinase 1/2. Significantly, low-level laser (LLL) irradiation increases NDR1 SUMOylation and subsequent p38/ERK1/2 activation via down-regulation of SENP2, leading to faster keratinocyte migration. Our findings fill the gaps that linger in the basic mechanisms underlying LLL therapy.-Xiao, N., Li, H., Yu, W., Gu, C., Fang, H., Peng, Y., Mao, H., Fang, Y., Ni, W., Yao, M. SUMO-specific protease 2 (SENP2) suppresses keratinocyte migration by targeting NDR1 for de-SUMOylation.

Background: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of G i protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin., Purpose: The aim of the present work was to evaluate the potential of a G i -protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages., Materials and Methods: Guanosine 5'- O -(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1β, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated., Results: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1β, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS., Conclusion: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The biogenesis and functionality of pattern recognition receptors (PRRs) are critical for robust plant immune responses. Here, we present methods to determine the N-glycosylation state and ligand-induced activity of these receptors for comparative quantitative analysis. These techniques can be used to identify mutants and chemical inhibitors affecting PRR biogenesis and functionality. When combined, these techniques can provide useful insights on biological processes necessary to synthesize a properly membrane-localized and ligand-responsive PRR.

Vertebrate β-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four β-catenin-related proteins present in the round worm Caenorhabditis elegans. The fourth β-catenin-related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription of Wnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein. We showed previously that WRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate β-catenin.

There is abounding evidence that neuroinflammation plays a major role in the pathogenesis of neurodegeneration and neuropathic pain. Chemokine-induced recruitment of peripheral immune cells is a central feature in inflammatory neurodegenerative disorders. Immune cells, glial cells and neurons constitute an integral network that coordinates the immune response by releasing inflammatory mediators that in turn modulate inflammation, neurodegeneration and the signal transduction of pain, via interaction with neurotransmitters and their receptors. The chemokine monocyte chemoattractant protein-1/ chemokine (C-C motif) ligand (MCP-1/CCL2) and its receptor C-C chemokine receptor (CCR2) play a major role in mediating neuroinflammation and targeting CCL2/CCR2 represents a promising strategy to limit neuroinflammation-induced neuropathy. In addition, the CCL2/CCR2 axis is also involved in mediating the pain response. Key cellular signaling events such as phosphorylation and subsequent activation of mitogen activated protein kinase (MAPK) p38 and its substrate MAPK-activated protein MAPKAP Kinase (MK) MK-2, regulate neuroinflammation, neuronal survival and synaptic activity. Further, MAPKs such as extracellular signal-regulated kinases (ERK), c-jun N-terminal kinase (JNK) and p38 play vital roles in mediating the pain signaling cascade and contribute to the maintenance of peripheral and central neuronal sensitization associated with chronic pain. This review outlines the rationale for developing therapeutic strategies against CCL2/CCR2 and MAPK signaling networks, identifying them as novel therapeutic targets for limiting neuroinflammation and neuropathic pain.

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis, including both energy intake and energy expenditure. It mediates the actions of a number of hormones on energy balance. The endogenous ligands for MC4R include peptide agonists derived from processing of proopiomelanocortin and the antagonist Agouti-related peptide (AgRP). Wild-type MC4R has some basal (constitutive) activity. Naturally occurring and laboratory-generated mutations have been identified, which results in either increased or decreased basal activities. Impaired basal signaling has been suggested to be a cause of dysregulated energy homeostasis and early-onset obesity, although several constitutively active mutations have also been identified from obese patients. AgRP and several small-molecule antagonists have been shown to be inverse agonists in the Gs-cAMP pathway. However, in the extracellular signal-regulated kinase (ERK) 1/2 pathway, we showed that these inverse agonists are potent agonists, demonstrating convincingly that they are biased ligands. We also showed that some mutations that do not cause constitutive activation in the Gs-cAMP pathway cause constitutive activation in the ERK1/2 pathway, suggesting that they are biased receptors. The physiological and potential pathophysiological relevance of the biased constitutive signaling in MC4R and therapeutic potential remain to be investigated., (© 2014 Elsevier Inc. All rights reserved.)

Receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca(2+) mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca(2+)/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca(2+) mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca(2+) mobilization induced by aluminum fluoride (AlF4 (-)), a G-protein activator, with or without RANKL and the effects of AlF4 (-) on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4 (-) induces intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations, which is dependent on extracellular Ca(2+) influx. Notably, co-stimulation of AlF4 (-) with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4 (-). Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca(2+)]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.

Oxycom applications increased plant growth and population levels of Meloidogyne incognita on susceptible tomato. A single Oxycom drench at 2,500 ppm applied 7 days prior to inoculation with M. incognita provided remediation of plant growth measured 63 days later. This occurred without reducing nematode population levels. Follow-up drenches at 2,500 ppm at 10-day intervals stunted shoots and roots (P = 0.05). The same application rates at 20-day intervals did not reduce plant growth. Plants receiving multiple drenches had more galls (P = 0.05), females, and second-stage juveniles (J2) per root system compared to plants receiving only the single treatment. Foliar mass and height of plants treated with a single pre-inoculation Oxycom drench were indistinguishable from plants without nematodes. Oxycom treatments activated signaling pathways for plant defense as confirmed by detection of elevated defense gene transcripts in root tissues. The finding of increased reproduction of root-knot nematode without loss of plant growth is consistent with the definition of induced tolerance. Frequency, rate, and timing of applications need further study with other nematodes and various field settings.