Dilated cardiomyopathy is the most frequent form of myocardial disease. Many factors contribute to dilated cardiomyopathy, for instance, long-term use of doxorubicin, one of the anthracyclines clinically used for cancer chemotherapy, result in dilated cardiomyopathy and congestive heart failure. However, the mechanism underlining doxorubicin-induced cardiomyocyte is still not fully understood. In this study, we evaluate the effects and their mechanisms of PPARα and PGC-1α pathways in doxorubicin induced mice cardiomyocytes. In vitro, cardiomyocytes isolated from hearts of adult FVB/NJ mice were treated with doxorubicin, GW 6471 (PPARα inhibitors) and WY14643 (PPARα agonists). The expression of PPARα and PGC-1α were detected via western blotting and Quantitative Real-Time PCR methods. Changes in energy and substrate metabolism were analyzed. MTT and flow cytometry were used for cell proliferation and apoptosis analysis. We detected expression of PPARα and PGC-1α was significantly higher in control group than doxorubicin group. Mitochondrial dysfunction was found in doxorubicin group including lower content of high-energy phosphates, significantly decreased mitochondrial ANT transport activity and markedly reduced mitochondrial membrane potential compared with control group. Metabolic remodeling existed in doxorubicin group because of higher concentration of free fatty acid and glucose consumption than of control group. More accumulations of reactive oxygen species were detected in doxorubicin group. The decreased cell viability and increased cell apoptosis observed in doxorubicin group. Severe apoptosis in doxorubicin group was verified by a set of markers including Bax, Bcl-2, cytosolic cytochrome c and caspase-3 up-regulation expression. These findings indicate that the PPARα and PGC-1α are closely involved in energy metabolism remodeling and apoptosis in cardiomyocytes.

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.

DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA.

Methamphetamine (METH) administration alters gene expression in the nucleus accumbens (NAc). We recently demonstrated that an acute METH injection produced prolonged increases in the expression of immediate early genes in the NAc of HDAC2-deficient mice, suggesting that HDAC2 might be an important regulator of gene expression in the rodent brain. Here, we tested the possibility that HDAC2 deletion might also impact METH-induced changes in the expression of various HDAC classes in the NAc. Wild-type (WT) and HDAC2 knockout (KO) mice were given a METH (20 mg/kg) injection, and NAc tissue was collected at 1, 2, and 8 h post treatment. We found that METH decreased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA expression but increased HDAC6 mRNA levels in the NAc of WT mice. In contrast, the METH injection increased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA levels in HDAC2KO mice. These observations suggest that METH may induce large-scale transcriptional changes in the NAc by regulating the expression of several HDACs, in part, via HDAC2-dependent mechanisms since some of the HDACs showed differential responses between the two genotypes. Our findings further implicate HDACs as potential novel therapeutic targets for neurotoxic complications associated with the abuse of certain psychostimulants.

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Alternatively activated macrophages are more frequently involved in tumor growth, angiogenesis, and immunosuppression. A previous study showed that paeoniflorin, the major active constituent of Paeonia lactiflora Pallas, can inhibit tumor growth and lung metastases of Lewis lung tumor-bearing mice. This study tried to investigate whether paeoniflorin inhibited lung cancer metastasis by inhibiting the alternative activation of macrophages (M2 macrophage). Using a viability assay, the cytotoxicity of paeoniflorin on Lewis lung cancer cells and peritoneal macrophages were investigated. In vitro scratch wound and in vivo lung metastasis experiments were used to test the ability to inhibit the migration of paeoniflorin and the function of M2 macrophages. Flow cytometry was performed to test the cell cycle of Lewis lung cancer cells, and to test the M2 macrophages in peritoneal macrophages and subcutaneous transplantable tumor. It was found that paeoniflorin showed no inhibitory effect on the growth of Lewis lung cancer cells and peritoneal macrophages of mouse in vitro. Paeoniflorin could attenuate the migration of LLC stimulated by alternatively activated macrophages (stimulated for 24 h and 48 h, paeoniflorin 1, 3, 10, 30, 100 μmol·L(-1), P < 0.01 or P < 0.05 vs control group). Paeoniflorin could decrease the cell populations at S phases (paeoniflorin 10, 30, 100 μmol·L(-1), P < 0.05 vs control group) and increase the cell populations at G0-G1 phases of Lewis lung cancer cells (paeoniflorin 100 μmol·L(-1), P < 0.05 vs control group) and reduce the numbers of M2 macrophages in peritoneal macrophages induced by IL-4 (paeoniflorin 1, 3, 10, 30, 100 μmol·L(-1), P < 0.01 vs Control group). Paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft and decrease the numbers of M2 macrophages in subcutaneous xenograft tumour in vivo (paeoniflorin 20, 40 mg·kg(-1), P < 0.01 vs control group). These results suggest that paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft partly through inhibiting the alternative activation of macrophages.

In the present study, we investigated anti-inflammatory effects of Sangxingtang (SXT) on acute lung injury using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The cell counting in the bronchoalveolar lavage fluid (BALF) was performed. The degree of lung edema was evaluated by measuring the wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activities were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were assayed by the enzyme-linked immunosorbent assay methods. Pathological changes of lung tissues were observed by Hematoxylin and eosin (HE) staining. The inflammatory signaling pathway-related proteins nuclear factor mitogen activated protein kinases (P38MAPK), extracellular regulated protein kinases (Erk), c-Jun N-terminal kinase (Jnk) and nuclear transcription factor (NF-κB) p65 expressions were measured by Western blotting. Our results showed that the treatment with the SXT markedly attenuated the inflammatory cell numbers in the BALF, decreased the levels of P-P38MAPK, P-Erk, P-Jnk and P-NF-κB p65 and the total protein levels in lungs, improved the SOD activity and inhibited the MPO activity. Histological studies demonstrated that SXT substantially reduced the LPS-induced neutrophils in lung tissues, compared with the untreated LPS group. In conclusion, our results indicated that SXT had protective effects on LPS-induced ALI in mice.

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.

Globally, food industries have made significant progress in order to increase the shelf-life of food products and have fewer economic losses. In this sense, the use of organomodified clays destined to be incorporated in polymer matrices play a novel role, leading to improved materials named nanocomposites with enhanced technological profiles. Due to the presence of these clays into the package, the safety of the consumers is a main concern. Cloisite(®)30B and Clay1 are two organomodified clays containing quaternary ammonium salts as modifiers, that can be potentially used to reinforce packaging polymers. Available toxicity data about these clays, specifically genotoxicity, is still limited and inconclusive in some aspects. Thus, the purpose of this work was to evaluate both clays ability to induce genomic instability through the cytokinesis block micronucleus cytome assay (CBMN) and for the first time, their influence in the modulation of several genes involved in genotoxicity and cell death mechanisms. Overall, no genotoxicity response was obtained in any case at the conditions tested. On the other hand, significant expression changes were observed on the genes selected. Nevertheless, further studies are highly needed to elucidate and increase the knowledge about the molecular mechanisms of clays toxicity.

Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor.

Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

Ras GTPase-activating proteins (GAPs) are important regulators for Ras activation, which is instrumental in tumor development. However, the mechanism underlying this regulation remains elusive. We demonstrate here that activated EGFR phosphorylates the Y593 residue of the protein known as family with sequence similarity 129, member B (FAM129B), which is overexpressed in many types of human cancer. FAM129B phosphorylation increased the interaction between FAM129B and Ras, resulting in reduced binding of p120-RasGAP to Ras. FAM129B phosphorylation promoted Ras activation, increasing ERK1/2- and PKM2-dependent β-catenin transactivation and leading to the enhanced glycolytic gene expression and the Warburg effect; promoting tumor cell proliferation and invasion; and supporting brain tumorigenesis. Our studies unearthed a novel and important mechanism underlying EGFR-mediated Ras activation in tumor development.

Solanum corymbiflorum is popularly known as "baga-de-veado" and its leaves are applied on inflamed legs, scabies, tick bite, boils, mastitis, low back pain and otitis. The aim of this study was evaluate anti-inflammatory in vivo activity and relate this activity with antioxidant compounds present in the extract of S. corymbiflorum leaves. The extract from S. corymbiflorum leaves topically applied was able to reduce the croton oil-induced ear edema and myeloperoxidase (MPO) activity with maximum inhibition of 87±3% and 45±7%, rescpectively in the dose of 1mg/ear. Similar results were found for positive control dexamethasone, which presented inhibitions of ear edema and MPO activity of 89±3% and 50±3%, respectively in a dose of 0.1mg/ear. These findings are due, at least in part, the presence of polyphenols (195.28mg GAE/g) and flavonoids, as chlorogenic acid (59.27mg/g), rutin (12.72mg/g), rosmarinic acid, caffeic acid and gallic acid found by high performance liquid chromatography (HPLC) analysis. This species showed potencial antioxidant by 1,1-diphenyl-2-picrylhydrazyl (DPPH), and carbonyl groups in proteins methods which may be related with the presence of this compounds. This species possess anti-inflammatory activity confirming their popular use for the local treatment of skin inflammatory disorders.

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to the nuclear receptor superfamily and it has received much attention because of its anti-inflammatory activity. However, the underlying molecular mechanism is not completely understood. In the present study, we demonstrated that the level of PPARγ is inversely correlated with that of high mobility group box 1 (HMGB1, a late proinflammatory mediator) in patients with sepsis. Activation of PPARγ inhibits the basal and LPS-induced expression of HMGB1. The PPARγ-mediated inhibition of HMGB1 is associated with the upregulation of miR-142-3p, which can target the 3'-UTR of HMGB1, by directly binding to the PPRE in the miR-142-3p promoter region. Functional experiments reveal that the PPARγ-induced miR-142-3p suppresses inflammatory response in vivo. These results suggest that PPARγ-mediated upregulation of miR-142-3p inhibits the HMGB1 expression, which, in turn, is a novel anti-inflammatory mechanism of PPARγ and has an important role in the treatment of inflammatory diseases.

The cellular mechanisms that control protein degradation may constitute a non-oncogenic cancer cell vulnerability and, therefore, a therapeutic target. Although this proposition is supported by the clinical success of proteasome inhibitors in some malignancies, most cancers are resistant to proteasome inhibition. The ATPase valosin-containing protein (VCP; p97) is an essential regulator of protein degradation in multiple pathways and has emerged as a target for cancer therapy. We found that pharmacological depletion of VCP enzymatic activity with mechanistically different inhibitors robustly induced proteotoxic stress in solid cancer and multiple myeloma cells, including cells that were insensitive, adapted, or clinically resistant to proteasome inhibition. VCP inhibition had an impact on two key regulators of protein synthesis, eukaryotic initiation factor 2α (eIF2α) and mechanistic target of rapamycin complex 1 (mTORC1), and attenuated global protein synthesis. However, a block on protein translation that was itself cytotoxic alleviated stress signaling and reduced cell death triggered by VCP inhibition. Some of the proteotoxic effects of VCP depletion depended on the eIF2α phosphatase, protein phosphatase 1 regulatory subunit 15A (PPP1R15A)/PP1c, but not on mTORC1, although there appeared to be cross-talk between them. Thus, cancer cell death following VCP inhibition was linked to inadequate fine-tuning of protein synthesis and activity of PPP1R15A/PP1c. VCP inhibitors also perturbed intracellular amino acid levels, activated eukaryotic translation initiation factor 2α kinase 4 (EIF2AK4), and enhanced cellular dependence on amino acid supplies, consistent with a failure of amino acid homeostasis. Many of the observed effects of VCP inhibition differed from the effects triggered by proteasome inhibition or by protein misfolding. Thus, depletion of VCP enzymatic activity triggers cancer cell death in part through inadequate regulation of protein synthesis and amino acid metabolism. The data provide novel insights into the maintenance of intracellular proteostasis by VCP and may have implications for the development of anti-cancer therapies.

Alzheimer's disease (AD) is the major cause of dementia worldwide. The pharmacological activation of nuclear receptors (Liver X receptors: LXRs or Retinoid X receptors: RXR) has been shown to induce overexpression of the ATP-Binding Cassette A1 (ABCA1) and Apolipoprotein E (ApoE), changes that are associated with improvement in cognition and reduction of amyloid beta pathology in amyloidogenic AD mouse models (i.e. APP, PS1: 2tg-AD). Here we investigated whether treatment with a specific LXR agonist has a measurable impact on the cognitive impairment in an amyloid and Tau AD mouse model (3xTg-AD: 12-months-old; three months treatment). The data suggests that the LXR agonist GW3965 is associated with increased expression of ApoE and ABCA1 in the hippocampus and cerebral cortex without a detectable reduction of the amyloid load. We also report that most cells overexpressing ApoE (86±12%) are neurons localized in the granular cell layer of the hippocampus and entorhinal cortex. In the GW3965 treated 3xTg-AD mice we also observed reduction in astrogliosis and increased number of stem and proliferating cells in the subgranular zone of the dentate gyrus. Additionally, we show that GW3965 rescued hippocampus long term synaptic plasticity, which had been disrupted by oligomeric amyloid beta peptides. The effect of GW3965 on synaptic function was protein synthesis dependent. Our findings identify alternative functional/molecular mechanisms by which LXR agonists may exert their potential benefits as a therapeutic strategy against AD.

A series of novel (E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety were firstly synthesized and their immunosuppressive activities were evaluated. Among all the compounds, 4n exhibited the most potent inhibitory activity (IC50 = 1.18 μM for lymph node cells and IC50 = 0.28 μM for PI3Kγ), which was comparable to that of positive control. Moreover, selected compounds were tested for their inhibitory activities against IL-6 released in ConA-simulated mouse lymph node cells, 4n exhibited the most potent inhibitory ability. Furthermore, in order to study the preliminary mechanism of the compounds with potent inhibitory activity, the RT-PCR experiment was performed to assay the effect of selected compounds on mRNA expression of IL-6. Among them, compound 4n strongly inhibited the expression of IL-6 mRNA.

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.

Breast cancer cells generally develop resistance to TNF-Related Apoptosis-Inducing Ligand (TRAIL) and, therefore, assistance from sensitizers is required. In our study, we have demonstrated that Spleen tyrosine kinase (Syk) inhibitor Bay 61-3606 was identified as a TRAIL sensitizer. Amplification of TRAIL-induced apoptosis by Bay 61-3606 was accompanied by the strong activation of Bak, caspases, and DNA fragmentation. In mechanism of action, Bay 61-3606 sensitized cells to TRAIL via two mechanisms regulating myeloid cell leukemia sequence-1 (Mcl-1). First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation. Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level. In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk. In summary, Bay 61-3606 downregulates Mcl-1 expression in breast cancer cells and sensitizes cancer cells to TRAIL-mediated apoptosis.