Month: September 2017

Recent evidence indicates that such fatty acids are partly, responsible for inducing steatosis and fueling hepatocyte insulin resistance . GLP-1 is an incretin that is released from the L-cells of the small intestine which targets pancreatic ?-cells to release insulin and reduce glucagon production in response to food intake . Insulin resistance also results in defective glucagon like peptide-1 release . Recently we demonstrated that GLP-1 receptors are present on human Tofacitinib hepatocytes and that exposure of hepatocytes to GLP-1 receptor agonists led to a reduction of fat load in hepatocyte cell lines, HepG2 and Huh7. Little work, however, has been performed in primary human hepatocytes in vitro to elucidate the lethal potential of such fatty acids discussed previously . Fatty acids are known to induce the unfolded protein response , a compensatory cellular mechanism to handle cell stress . This process, including protein degradation and inhibition of translation allows cells to survive stress caused by defective proteins. In addition to the UPR an additional component to maintain a healthy proteome in the cell – lysosomal degradation or autophagy, has been shown to be critical in removing potentially toxic fatty acids from cells. In addition to defective protein degradation, lysosomes have also been shown to degrade other intracellular components, including whole organelles, lipid deposits, proteinaceous inclusions and aggregates . Singh et al. recently demonstrated that a fatty acid load in mouse hepatocytes is reduced by macroautophagy . Another type of autophagy, chaperone mediated autophagy has also been demonstrated to reduce cell stress by removing proteins that have a signal sequence . While the molecular details regarding chaperone mediated autophagy are less well-developed regulators of CMA have recently been identified in murine hepatocytes .

Identified genes are involved in a wide range of cellular processes suggesting new and expanded roles for this transcription factor. As detailed above, Arx directly regulates genes required for R428 Axl inhibitor cell-cycle control, tight-junction dynamics, cell morphology, neuronal migration and differentiation as well as synaptic plasticity modulation, neurotransmission and axonal guidance. Among the different targets we identified, a number of genes are known to be important for brain development and some have already been linked to human disorders. But in addition, we identified new genes which may be good candidates to test in human neurological and psychological diseases. Further studies to understand their function and their relation to Arx will certainly bring new insight into the understanding of the pathophysiology of intellectual disability and epilepsy. Chromatin immunoprecipitation on embryonic brains from wild-type mice was performed following a similar protocol. All animal procedures were performed in accordance with French and international guidelines and were approved by the French review board ministe`re . Briefly, adult pregnant female mice were killed by cervical dislocation and brains were extracted from day 15.5 embryos in cold PBS. Following brain isolation, the whole forebrain including ventral telencephalon, thalamus, cerebral cortex and olfactory bulbs were frozen in liquid nitrogen and kept at 280uC for further experiments. For each experiment, tissue was pooled from 3 embryos. Tissue disaggregation and homogenization were performed in liquid nitrogen using a pestle and a mortar. Samples were then transferred into a 15 ml Falcon tube and fixed in a solution containing PBS, 1% formaldehyde and Protease Inhibitor Cocktail . Following steps were identical to those described above. Intensity data on each array were normalized with the Lowess method , pooled and represented on a graph. To identify regions of significant Arx association, the enrichment of each probe on the array was calculated as the log2-ratio of the intensities of Arx-immunoprecipitated DNA to control input chromatin . One first important assumption is that points corresponding to non positive probes are distributed symmetrically around the axis x= y.

We therefore examined whether activation of STAT3 via IL-6 stimulation led to repression of Necdin expression in the prostate cancer cell lines DU145 and PC3. These cell lines harbor low levels of constitutively active STAT3 , which can be further induced by stimulation with IL-6. Cells were serum starved for 3 h prior to treatment with IL-6 for 12 or 24 h. Total protein was prepared and analyzed by Western blot. Figure 4A shows that IL-6 stimulation resulted in increased STAT3 activity within the cells and demonstrated corresponding down-regulation of Necdin expression upon IL-6 stimulation, in both cell lines. This confirms that IL-6 is capable of repressing Necdin expression via STAT3 in prostate cancer cells. Since EGFR and Src signaling pathways contribute to STAT3 activation in breast cancers , we evaluated Necdin expression levels in human breast cancer cell lines with different levels of endogenous STAT3 activity. Figure 4B shows that p- STAT3 protein levels were high in MDA-MB-468 cells, slightly lower in MDA-MB-231 and very low in MCF-7 cells. Necdin protein expression inversely correlated with p-STAT3 levels, being expressed at a low level in MDA-MB-468 and MDA-MB-231 cells, but exhibited much CP-358774 higher expression in MCF-7 cells. To test the hypothesis that constitutively activated STAT3 has a causal role in suppressing Necdin expression in tumor cells, we examined whether transient activation of STAT3 signaling could down-regulate Necdin expression. MCF7 cells express high levels of Necdin , however when transiently transfected with v-Src or STAT3-C for 48 h, Necdin protein expression is inhibited. This demonstrates that even a transient 2- fold increase in STAT3 activation in these cells is sufficient to effectively repress the expression of Necdin . The transcriptional profile of a cell expressing constitutivelyactive STAT3 is predicted to be very different compared to a cell where STAT3 is under tight regulation. Our initial hypothesis was that STAT3 promotes widespread changes in global gene expression patterns, including both direct and indirect targets. We took a broad approach by studying global gene expression changes using microarray analysis in cells expressing constitutively- activated STAT3.

The common neuropathological hallmarks in neurons and glia are microscopic proteinaceous inclusions, composed mainly of aggregated fibrillar alpha-synuclein . aSN is an abundant presynaptic protein in the brain. Its 140 amino-acid sequence is highly homologous across human, rat and mouse . Initially, aSN microscopic aggregates were postulated to play a key role in the pathophysiology of a-synucleinopathies. Neurotoxicity findings implicate aSN protofibrils, soluble aSN protein WZ-4002 EGFR/HER2 inhibitor complexes, posttranslationally modified forms of aSN , phosphorylated at serine 129 , as well as mono- and di-ubiquitinated aSN forms . In DLB brains more than 90% of the insoluble aSN is phosphorylated at Ser129 compared to about 4% phosphorylated at Ser129 in brains of normal individuals. Furthermore, Ser129 phosphorylated aSN is targeted to mono- and di-ubiquitination in a-synucleinopathy brains . Extensive phosphorylation at Ser129 and/or its mono- and di-ubiquitination are critical events in the pathophysiology of aSN. However, direct experimental evidence supporting this notion is lacking and it is still debated whether these molecular forms of aSN are on the critical pathophysiological path rather than representing molecular epiphenomena of the disease process. As multiple toxic mechanism have been proposed for aSN, it is important to determine which of its molecular forms are on the critical pathophysiological path. One main hypothesis of aSN toxicity is based on its capability to form toxic oligomers. Familial forms of Parkinson��s disease possess mutant forms of aSN A53T and A30P that form oligomers more rapidly than wildtype aSN. In idiopathic forms of a-synucleinopathies that lack heritable aSN mutations, it is speculated that compromised handling of aSN and/or specifically modified forms are hampering aSN catabolism as well as that of other proteins. Oxidative damage of aSN could change aSN into toxic forms that trigger such a pathophysiological cascade . It is unclear how critical to the disease process are some of the differences in aSN amino-acid sequence between human, rat and mouse. There is no solid evidence for endogenous mouse aSN coaggregating with human aSN expressed in transgenic rodent models .

Importantly, both glutamines of the Q loop of the D1 domain of DLAR were seen to cluster with the R loop when the D1 domain was present in isolation. This interaction was uncoupled in the presence of the D2 domain of DLAR where one of the glutamines now clustered separately. This could perhaps account for the decrease in the activity of the D1 domain of DLAR in the presence of its D2 domain as it disrupts the glutamine network with the active site residues. Interestingly, in the case of PTP99A, the residues of the WPD loop formed a separate cluster from the active site when the D1 domain was present alone. This WPD loop cluster was seen to be merged with the active site residues in the presence of the D2 domain. It thus appears likely that the D2 domain of PTP99A enhances the activity of its D1 domain by strengthening the interaction networks between the active site residues and the WPD loop. Differences in the functional roles of RPTPs have often been explained by sequence-structure variations as well as spatiotemporal effects in developmental processes. The role of extracellular domains of these RPTPs is clear from unambiguous genetic data – deletions in the Immunoglobulin�Clike domains of DLAR are lethal, while deletions in the Fibronectin type III repeats are not. The Fibronectin type III repeats are essential for Drosophila oogenesis suggesting that these domains are used in distinct signaling pathways and cell fate decisions in Drosophila development . While the extracellular domains of these RPTPs are required for their proper localization in the nerve cell membrane, the signaling pathways at the growing axon cone are coordinated by the concerted activity of their cytosolic PTP domains. The tandem PTP domains of double domain RPTPs form an interesting model system. In particular, the role of the catalytic D2 domain in the function of these proteins is unclear from genetic data. For example, the D1 domains of DLAR and DPTP69D have been examined for their ability to rescue the homozygous deletion mutations of these genes. In the case of DLAR, D1 was found to be redundant as D2 could itself partially rescue the DLAR 2/2 phenotype . In the case of DPTP69D however, the active D1 domain was essential to rescue the DPTP69D 2/2 lethality . These contradictory findings suggest a complex PD-0325901 biological activity interplay between the PTP domains when attached in tandem. A combination of biochemical studies using activity measurements, protein-substrate interactions and MD simulations were performed to understand the molecular basis of modulation of phosphatase activity in the two tandem PTP domains of DLAR and PTP99A.