Bioactive Screening Library

People who binge-eat show altered prefrontal-corticalstriatal-insula responses to appetitive stimuli. One fMRI study compared neural activation in women with AN and BN who passively viewed images of food, finding reduced prefrontal cortex but greater cerebellum activation in women with BN. However, no fMRI study to date has attempted to deliberately evoke cognitions associatied with eating in women with AN and BN, and to measure neural responses to food images. The aim of this exploratory study, which uses a novel paradigm whereby eating-related cognitions are evoked by thinking about eating food shown in images, is to demonstrate a differential pattern of activation in cortico-striatal-insula regions between women currently ill with AN and BN. We hypothesize that: a) women with AN and BN compared to healthy controls have reduced striatal-insular cortex, and greater prefrontal cortical activation when thinking about eating food versus non-food items shown in images; b) women with BN have reduced prefrontal cortical activation but greater striatal-insular cortex responses in comparison to women with AN. Using a novel fMRI paradigm, whereby participants thought about eating food shown in images rather than passively viewing them, we have compared neural responses in women with BN and AN. These exploratory data provide the first evidence that patterns of food consumption combined with degree of restraint over eating behaviour may alter how the brain responds when thinking about eating food. However, caution must be exercised; despite stringent threshold corrections, the small sample sizes make these intriguing results somewhat tentative at this stage, and in need of further clarification with larger cohorts. Activation of the dorsolateral prefrontal cortex, visual cortex and cerebellum in response to food versus non-food images was found in women with AN and BN. Additionally, women with BN show greater somatosensory and motor responses in the right insular cortex and post-central gyrus. Women with BN showed increased activation in somatosensory, motor and appetitive regions in comparison to AN and HC. Furthermore, women with BPAN had reduced activation in comparison to women with BN in regions associated with appetitive and motor responses. Thus, although women with BPAN partake in binge eating and purging behaviour in a AZD6244 similar way to women with BN, their heightened control over eating may be achieved by a reduced drive to eat over the long term. Thus, these results suggest a clear BN-specific map of brain activation in response to thinking about eating food shown in images, which is primarily characterized by signs of increased appetitive, somatosensory and motor responses, in parallel with activation of prefrontal cortex cognitive inhibition. However, these intriguing suggestions need to be tested further.

In order to summarize the results obtained in this study we have build a hypothetical model-diagram of the EGF/IGF-2 regulatory circuit functioning during the transition from transgenic to tumor state. This model combines two signal NVP-BEZ235 transduction networks of the signal flow from EGF and IGF-2 reaching a number of transcription factors, that in turn, regulate expression of several important genes that encode components of the upstream network. Thus created feedback loops should play an important role in emerging as well as in stabilizing the cancer state of the cells, In this model, we can propose multiple paths of signals initially coming massively from EGF in the transgenic cells and triggering activity of several TFs, such as C/EBP-alpha, GR and HNF4alpha, that down-regulate expression of their target gene encoding EGF as well as Cav1, thus trying to compensate the excess of the EGF stimulus in the cell. At the same time, through parallel signaling cascades and activation of a number of other TFs, such as c-Ets-1, PPAR-gamma, STAT family factors, cMyb and others, upregulation of expression of Igf2 gene as well as Igfbp6 and Pparg can be achieved. Due, to several feedback loops on different levels of the network coming from these genes, we can speculate that a steady signal for upregulation of the Igf2 gene leads eventually to a sharp elevation of its expression with the consequence of increase of mitogenic activity of the cells, which marks the transition to the carcinogenic state. It was reported previously, that Igf2 gene is located in an imprinted area of genome and is repressed in most of tissues of the adult organism. Loss of imprinting of the Igf2 gene is one of the most common observations in cancers. It was shown that the imprinting status is maintained by binding of CTCF repressor to an intergenic area of the Igf2 gene and loss of this binding can lead to 10-fold elevation of Igf2 expression. We propose a model where the feedback mechanisms involved in the Igf2 epigenetic control through multiple transcription factors, activators and repressors, play the major role in the switching the cells to malignant transformation. In conclusion, promoter analysis of the differentially expressed genes enabled us to identify transcription factor binding sites. Such integration of sequence information into signal transduction networks enabled an identification of key nodes upstream of the identified transcription factors. By searching for pairs of TF sites and integration of this information into the network analysis robust information can be retrieved in an unbiased manner that clearly identifies keynodes and molecules acting in concert in defined biological conditions. Therefore, we propose a sequence of events whereby the insulin-like growth factor pathway represents an important molecular switch in malignantly transformed liver cells.

In 5 of 6 foreground sets, POU motifs were more strongly associated with the transgenic state than with the tumor state. This difference was most pronounced in analyses of downregulated and PI-103 specific downregulated gene sets, where dots representing POU matrices are located far away from the mass of points. Notably, sites of some POU motifs were also more than 2- fold enriched in promoters of downregulated genes in tumor. Oct1 matrices were the top ranked motifs in downregulated and specific downregulated genes in tumor. However, promoters of upregulated genes were detectably enriched with POU sites in the transgenic state only. These results suggest that POU factors contributed to the switch from transgenic to tumor state. Furthermore, their activity may represent a major cause of observed downregulation events. Among the POU transcription factors represented by matrices identified in the analyses, the expression profile of Pou5f1 resembled well the observed progression-state specific enrichment of its binding sites. The Pou5f1 gene exhibited a high fold change in the transgenic state, which had decreased in the tumor state. Indeed, the Pou5f1 gene is specifically expressed in embryonic stem cells and in tumor cells, but not in cells of differentiated tissues. Transcription factors with a Forkhead domain also showed association with the transgenic state. This signal was best observed in upregulation and cell cycle gene sets, yet subtle enrichment in transgenic promoters could also be detected in specific downregulation and specific upregulation, where the FREAC2 motif ranked among the top 15 PWMs. In the upregulation set, Foxd3 binding sites showed the strongest signal after Oct1 sites. This would support a potential role of Oct4, as corepression through overlapping binding sites of Oct4 and Foxd3 was previously reported. According to expression measurements, Foxd3 was potentially downregulated in both progression states, although measured expression differences were not statistically significant. Instead, Foxc1 expression parallels the stronger enrichment of Forkhead binding sites in transgenic promoter sets, as it is specifically upregulated in the early progression state. Promoters of upregulated genes in tumor were associated with binding sites of cell cycle regulators such as AP1-like factors, STAT, and E2f, of which Atf3, Jun, and E2f3 were significantly upregulated in both transgenic and tumor cells. This finding supports the stronger regulation of cell cycle processes in tumor detected by comparative GO analysis. The analysis of cell cycle gene promoters suggests E2f factors as the most important regulators in both states, whereas a tendency towards higher q-values in the tumor set was observed for several E2f motifs. Notably, the Myc-associated zinc finger protein was detected in the transgenic cell cycle gene set.

A glaucomaassociated mutant of optineurin shows reduced binding to CYLD. This mutant, unlike wild type optineurin, does not inhibit Z-VAD-FMK 187389-52-2 TNFa-induced NF-kB activation. Optineurin is essential both for inhibition of TNFa-induced NF-kB activation by CYLD and its association with RIP. In addition we show that optineurin is required for CYLD mediated deubiquitnation of RIP. Our results thus show that the interaction of optineurin with CYLD is important for the regulation of TNFa-induced NF-kB activity. Ubiquitin binding proteins, with their diverse range of UBDs, have emerged as key regulators of NF-kB signalling. Optineurin was shown to inhibit TNFa-induced NF-kB activation by competing with NEMO for the binding of ubiquitinated RIP. However, our results suggest that the regulation of NF-kB activation by optineurin is more complex and binding of ubiquitinated RIP to UBD of optineurin is only one of the steps in this complex regulation. Although ubiquitinated RIP is known to be a substrate of CYLD, the mechanism by which CYLD is recruited to RIP to deubiquitinate it, is not clear. The results presented in this manuscript suggest that optineurin mediates interaction of deubiquitinase CYLD with polyubiquitinated RIP and this interaction is essential for deubiquitination of RIP by CYLD. Thus an important function of optineurin in the regulation of NF-kB signalling is to act as an adaptor protein bringing CYLD and its substrate RIP together to facilitate deubiquitination of ubiquitinated RIP by CYLD. CYLD targets multiple players of the NF-kB signalling pathway. How CYLD recognises and is targeted to a specific substrate like RIP is not well understood. CYLD interacts directly with some of its substrates like TRAF2 and NEMO. Emerging evidence suggests that CYLD might indirectly associate with some of its substrates through intermediary adaptor proteins. It has been shown that p62/sequestosome1 binds to TRAF6 through its UBD and recruits CYLD to TRAF6 to regulate its ubiquitination. An adaptor protein should be able to specifically bind to substrates polyubiquitinated with Lys63-linked ubiquitin chains since CYLD specifically deconjugates Lys63-linked polyubiqitin chains. In addition, such an adaptor protein should also interact with CYLD. Since optineurin interacts with Lys63-linked polyubiquitin chains through its UBD and also with CYLD, it is ideally suited to act as an adaptor for CYLD to recognise its substrates. The ABIN proteins with their homologous UBD similarly act as adaptors for the recruitment of A20 to its targets like NEMO. TNFa stimulus triggers assembly of Lys63-linked polyubiquitin chains on RIP which initially binds to NEMO to activate IKK complex. Optineurin binds to polyubiquitinated RIP through its UBD and recruits CYLD; this may prevent association of NEMO with RIP. Close proximity of binding sites of both CYLD and RIP on optineurin provides support for the adaptor function of optineurin in facilitating interaction of RIP and CYLD.

This results in the activation of IkB kinase complex consisting of the catalytic IKKa and b subunits and the regulatory subunit IKK-c/NEMO. IKK activation involves conjugation of Lys63-linked polyubiquitin chains to NEMO and its upstream regulators like RIP. RIP has emerged as a central adaptor in the pathways leading to IKK and NF-kB activation and also cell death. Following TNFa stimulation RIP is recruited to TNFR1 signalling complex and is rapidly ubiquitinated with Lys63-linked polyubiquitin chains. NEMO binds to polyubiquitinated RIP through its ubiquitin binding domain resulting in the activation of catalytic subunits of IKK. The recognition and association of ubiquitinated RIP with NEMO is essential for IKK activation. The activated catalytic subunits of IKK then phosphorylate IkB triggering its ubiquitination and degradation leading to nuclear translocation and activation of NF-kB. Given its role in diverse cellular processes, the activation of NF-kB is governed by several positive and negative regulators. With the increasing roleof ubiquitination, deubiquitinases like CYLD and A20 have emerged as key negative regulators of NF-kB activation. CYLD was originally identified as a tumor suppressor gene mutated in familial cylindromas. It is the first deubiquitinase shown to inhibit IKK activation. CYLD specifically catalyses cleavage of Lys63-linked polyubiquitin chains from its target proteins like RIP, NEMO and TRAFs to prevent NF-kB activation. Though CYLD targets multiple NF-kB signalling molecules, the mechanism by which CYLD recognises its substrate RIP to regulate NF-kB activation is not completely understood. Optineurin was recently identified as a negative regulator of NF-kB signalling whose expression is governed by NF-kB. It is a multifunctional protein involved in membrane trafficking, signal transduction, anti-viral responses and gene expression. The C-terminal region of optineurin has a novel bipartite UBD which shows homology with NEMO and ABIN1. This UBD of Optineurin, like NEMO, preferentially binds to Lys63-linked ubiquitin chains and does not show significant binding to Lys48-linked polyubiquitin chains. It was suggested that optineurin binds to polyubiquitinated RIP through its UBD to prevent association of NEMO with RIP, thus inhibiting NF-kB activation. Optineurin was identified as a gene mutated in certain glaucomas, a group of neurodegenerative eye diseases that cause blindness, and recently in familial amyotrophic lateral sclerosis. However, the nature of functional defects caused by mutations in optineurin is beginning to be BKM120 abmole bioscience understood only now. Recently we have identified CYLD as an interacting protein of optineurin in a yeast-two hybrid screen. This was reported briefly in a review without showing any data. However the functional significance of this interaction is not known. Since optineurin interacts with CYLD, the role of optineurin in the regulation of NF-kB signalling is likely to be complex.