Intracellular Phosphorylation Research Articles

Development of new promising antimetabolite, DFP-11207 with self-controlled toxicity in rodents

To reduce 5-fluorouracil (5-FU)-induced serious toxicities without loss of antitumor activity, we have developed DFP-11207, a novel fluoropyrimidine, which consists of 1-ethoxymethyl-5-fluorouracil (EM-FU; a precursor form of 5-FU), 5-chloro-2,4-dihydroxypyridine (CDHP; an inhibitor of 5-FU degradation), and citrazinic acid (CTA; an inhibitor of 5-FU phosphorylation). In vitro studies of DFP-11207 indicated that it strongly inhibited the degradation of 5-FU by dihydropyrimidine dehydrogenase (DPD) in homogenates of the rat liver, and also inhibited the phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT) in tumor tissues in a similar magnitude of potency by CDHP and CTA, respectively. Especially, DFP-11207 inhibited the intracellular phosphorylation of 5-FU in tumor cells in a dose-dependent manner whereas CTA alone did not protect intracellular 5-FU phosphorylation. These results postulate that DFP-11207 rapidly entered into the cell and the free CTA produced from DFP-11207 inhibited the phosphorylation of 5-FU in the cell. Furthermore, following oral administration of DFP-11207, CTA was found to be highly retained in the gastrointestinal (GI) tract compared to other tissues in rats. Interestingly, EM-FU, the prodrug of 5-FU was found to specifically produce 5-FU by various species of liver microsomes. When DFP-11207 was administered to rats, the plasma level of 5-FU was persisted for a long-time with lower Cmax and longer half-life than that from other 5-FU prodrugs. The antitumor activity of DFP-11207 was evaluated in human tumor xenografts in nude rats and found that DFP-11207 showed an antitumor activity in a dose-dependent fashion and its efficacy is equivalent to reference 5-FU drugs. In striking contrast, DFP-11207 manifested no or less 5-FU-related toxicities, such as a decrease in body weights, GI injury, and myelosuppression, especially thrombocytopenia. Taken together, the preclinical evaluation of DFP-11207 strongly indicates that DFP-11207 be a potential new version of the oral fluoropyrimidine prodrug for further clinical development.

Recombinant Human Thioredoxin-1 Protects Macrophages from Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation and Cell Apoptosis.

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway.

Valsartan (VAL), an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO). In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs). Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L) were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L), VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L), and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L) groups. The NO content in the VAL-treated HUVEC line (EA.hy926) was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05) and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.

Regulation of Human CD27+IgM+ B Cell Activation by Cis-Binding of the Inhibitory Molecule CD22 to CD22 Ligand (CD22L)

Background: ABO-incompatible (ABOi) heart transplantation (HTx) can be performed safely in infants and results in B cell tolerance to donor ABO antigens by mechanisms that remain unclear. We showed that the inhibitory molecule CD22 is more highly expressed on splenic CD27+IgM+ B cells, which contain the vast majority of ABO-specific antibody secreting cell precursors, compared to other splenic B cell subsets. CD22 expression was highest on infant CD27+IgM+ B cells and decreased with age. Binding of CD22 to sialic acids (CD22L) on the same cell (cis-binding) or different cells (trans-binding) regulates B cell activation and may lead to B cell tolerance. Here, we studied whether CD22-CD22L cis-binding affects activation of CD27+IgM+ and CD27-IgM+ B cells from pediatric and adult spleen. Methods: CD27+IgM+ and CD27-IgM+ B cells were isolated via fluorescence-activated cell sorting from pediatric (n=3; age range: 0–2.5 yr) and adult (n=3; age range: 21–46 yr) spleen obtained from organ donors. To ‘break’ CD22-CD22L cis-binding, sorted B cells were treated with neuraminidase (NEU) to cleave surface sialic acids. Activation of B cells was defined by analyzing intracellular phosphorylation of PLCγ2 (pY759) after stimulation with anti-IgM antibodies using ‘phospho-flow’ cytometry. Results: Kinetics of pY759 signaling after stimulation were similar between untreated CD27+IgM+ and CD27-IgM+ B cells as well as pediatric and adult samples. At peak response (1–3 min after stimulation), the median fluorescence intensity (MFI) of pY759 in pediatric CD27+IgM+ B cells was comparable to that in pediatric CD27-IgM+ B cells (350–614 vs. 259–565), whereas in adult cells the MFI was higher in CD27+IgM+ B cells than in CD27-IgM+ B cells (540–912 vs. 248–328). NEU treatment had a strong effect on kinetics and magnitude of pY759 signaling in both pediatric and adult CD27+IgM+ B cells: the MFI of pY759 increased to 506–2023 and 1086–2051, respectively, at peak response (4–8 min after stimulation). In contrast, treatment had minimal effect on pY759 signaling in CD27-IgM+ B cells. Conclusion: NEU treatment increased the magnitude of pY759 signaling in stimulated CD27+IgM+ B cells, but not CD27-IgM+ B cells, suggesting that CD22-CD22L cis-binding may play a role in control of activation of CD27+IgM+ B cells. This finding may contribute to unraveling mechanisms of B cell tolerance in ABOi HTx in children. Canadian National Transplant Research Program (CNTRP) investigator.

Distinct Expression and Function of FcεRII in Human B Cells and Monocytes.

FcεRII is a multifunctional low-affinity IgER that is involved in the pathogenesis of allergic, inflammatory, and neoplastic diseases. Although discrepancies in FcεRII-mediated functions are being increasingly recognized, the consequences of FcεRII activation are not completely understood. In this study, we evaluated the expression of FcεRII on human blood cells and found that it was primarily expressed on monocytes and B cells. Although IL-4 promoted expression of the FcεRIIb isoform on B cells and monocytes, the expression of the FcεRIIa isoform was not dependent on IL-4. Furthermore, FcεRII predominantly bound allergen-IgE complexes on B cells but not on monocytes. FcεRII-mediated allergen-IgE complex uptake by B cells directed Ags to MHC class II-rich compartments. FcεRII-bearing monocytes and B cells expressed high levels of the FcεRII sheddase a disintegrin and metalloproteinase 10, which implies that they are important sources of soluble FcεRII. Moreover, we identified that IgE immune complex stimulation of FcεRII activated intracellular tyrosine phosphorylation via Syk in B cells but not in monocytes. Importantly, FcεRII-mediated signaling by allergen-IgE immune complexes increased IFN-γ production in B cells of allergic patients during the build-up phase of allergen-specific immunotherapy. Together, our results demonstrate that FcεRII mediates cell type-dependent function in allergic reactions. In addition, the results identify a novel allergen-IgE complex/FcεRII/Syk/IFN-γ pathway in allergic responses and suggest that FcεRII may play a role in regulating allergic reactions via modulating IFN-γ production in B cells.

Pro-angiogenic Role of Insulin: From Physiology to Pathology.

The underlying molecular mechanisms involve in the regulation of the angiogenic process by insulin are not well understood. In this review article, we aim to describe the role of insulin and insulin receptor activation on the control of angiogenesis and how these mechanisms can be deregulated in human diseases. Functional expression of insulin receptors and their signaling pathways has been described on endothelial cells and pericytes, both of the main cells involved in vessel formation and maturation. Consequently, insulin has been shown to regulate endothelial cell migration, proliferation, and in vitro tubular structure formation through binding to its receptors and activation of intracellular phosphorylation cascades. Furthermore, insulin-mediated pro-angiogenic state is potentiated by generation of vascular growth factors, such as the vascular endothelial growth factor, produced by endothelial cells. Additionally, diseases such as insulin resistance, obesity, diabetes, and cancer may be associated with the deregulation of insulin-mediated angiogenesis. Despite this knowledge, the underlying molecular mechanisms need to be elucidated in order to provide new insights into the role of insulin on angiogenesis.

Epigenetic modifications influence TLR3 expression and activity

BackgroundToll‐like receptors (TLRs) are a family of pattern recognition receptors (PRRs) which recognise conserved pathogen associated molecular patterns (PAMPs) present in bacteria and viruses e.g. TLR3 recognises dsRNA from viruses. TLRs are expressed on a variety of cells including intestinal epithelial cells and play an important role in the immune response by stimulating cytokine release via phosphorylation of signalling proteins, but it is not known how their expression is regulated. We hypothesised that epigenetic mechanisms such as DNA methylation and histone deacetylation may play a role. In order to investigate this, we studied the effect of inhibition and/or genetic knockout of the enzymes responsible for these events (DNA methyltransferases (DNMT) or histone deacetylase complexes (HDAC)) on TLR3 expression and signalling in HCT‐116 intestinal epithelial cells.MethodsHCT‐116 (both wild‐type (WT) and DNMT 1/3B double knockout (DKO) cells) cells were either untreated or treated with the pan‐HDAC inhibitor suberanilohydroxamic acid (SAHA, 10μM, 48hrs), or the DNMT inhibitor 5‐aza‐2‐deoxycytidine (500nM, 72hrs), followed by stimulation with the TLR3 ligand Poly I:C (10μg/ml, 6 or 24 hrs). Cytokine mRNA expression was measured using quantitative polymerase chain reaction (QPCR) while protein release was measured using ELISA of the cell culture supernatant. Intracellular signalling protein phosphorylation was measured using Western blotting. Statistical analyses was performed in SPSS and in all cases n ≥ 3.ResultsTreatment of HCT‐116 WT cells with Poly I:C resulted in a significant increase in TLR3 expression (p&lt;0.001) and in the mRNA expression of cytokines (p&lt;0.001) and also the release of cytokines (p&lt;0.001). Poly I:C treatment also resulted in increased phosphorylation of signalling proteins (NF‐κB, IκBα, JNK, p38, IRF3). Pre‐treatment with SAHA, 5‐aza‐2‐deoxycytidine and genetic knockout of DNTM1/3b prevented this Poly I:C induced increase in TLR3 expression and cytokine mRNA expression, cytokine release and signalling protein phosphorylation.ConclusionInhibition of HDACs or DNMTs by pharmacological means or genetic knockout prevents TLR3 signalling and the activation of the TLR3 inflammatory pathway. This suggests that viral induced inflammation may be controlled using epigenetic modifying drugs.Support or Funding InformationCollege of Science Scholarship, NUI Galway

Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs)

The serine/arginine-rich protein kinases (SRPKs) have frequently been found with altered activity in a number of cancers, suggesting they could serve as potential therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-two trifluoromethyl arylamides based on the known SRPKs inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds 24, 30, and 36 presented IC50 values ranging between 6.0 and 35.7 μM. In addition, these three compounds were able to trigger apoptosis and autophagy, and to exhibit synergistic effects with the chemotherapeutic agent vincristine. Furthermore, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms. Therefore, novel compounds with increased intracellular effects against SRPK activity were obtained, contributing to medicinal chemistry efforts towards the development of new anticancer agents.

Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that is a major therapeutic target for the treatment of type 2 diabetes. Activation of this receptor promotes insulin secretion and blood glucose regulation. The GLP-1R can initiate signaling through several intracellular pathways upon activation by GLP-1. GLP-1R ligands that preferentially stimulate subsets among the natural signaling pathways (“biased agonists”) could be useful as tools for elucidating the consequences of specific pathways and might engender therapeutic agents with tailored effects. Using HEK-293 cells recombinantly expressing human GLP-1R, we have previously reported that backbone modification of GLP-1, via replacement of selected α-amino acid residues with β-amino acid residues, generates GLP-1 analogues with distinctive preferences for promoting G protein activation versus β-arrestin recruitment. Here, we have explored the influence of cell background across these two parameters and expanded our analysis to include affinity and other key signaling pathways (intracellular calcium mobilization and ERK phosphorylation) using recombinant human GLP-1R expressed in a CHO cell background, which has been used extensively to demonstrate biased agonism of GLP-1R ligands. The new data indicate that α/β-peptide analogues of GLP-1 exhibit a range of distinct bias profiles relative to GLP-1 and that broad assessment of signaling endpoints is required to reveal the spectrum of behavior of modified peptides. These results support the view that backbone modification via α→β amino acid replacement can enable rapid discovery of peptide hormone analogues that display substantial signal bias at a cognate GPCR.

T Cell Receptor Clustering - A Mechanism of Signal Transduction

Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. We use single-molecule localization microscopy and novel analysis to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and non-triggered TCR-CD3 complexes. For example, we found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. In summary, we propose a model in whcih TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Gene Expression Profiles of Human Phosphotyrosine Phosphatases Consequent to Th1 Polarisation and Effector Function.

Phosphotyrosine phosphatases (PTPs) constitute a complex family of enzymes that control the balance of intracellular phosphorylation levels to allow cell responses while avoiding the development of diseases. Despite the relevance of CD4 T cell polarisation and effector function in human autoimmune diseases, the expression profile of PTPs during T helper polarisation and restimulation at inflammatory sites has not been assessed. Here, a systematic analysis of the expression profile of PTPs has been carried out during Th1-polarising conditions and upon PKC activation and intracellular raise of Ca2+ in effector cells. Changes in gene expression levels suggest a previously nonnoted regulatory role of several PTPs in Th1 polarisation and effector function. A substantial change in the spatial compartmentalisation of ERK during T cell responses is proposed based on changes in the dose of cytoplasmic and nuclear MAPK phosphatases. Our study also suggests a regulatory role of autoimmune-related PTPs in controlling T helper polarisation in humans. We expect that those PTPs that regulate T helper polarisation will constitute potential targets for intervening CD4 T cell immune responses in order to generate new therapies for the treatment of autoimmune diseases.

STAT6 and STAT1 Pathway Activation in Circulating Lymphocytes and Monocytes as Predictor of Treatment Response in Rheumatoid Arthritis.

ObjectiveTo find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA).Methods19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-γ, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test.ResultsAt baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-γ -stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042).ConclusionCytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively. Both fulvestrant-resistant cell lines lost sensitivity to estrogen and anti-estrogens. We observed diminished ERα expression at both the protein and mRNA levels. To address the mechanism of gene expression regulation, we examined epigenetic alteration, especially the DNA methylation level of ERα gene promoters. MFR cells displayed high methylation levels upstream of the ERα gene, whereas no change in DNA methylation was observed in TFR cells. Hence, we examined the gene expression plasticity of ERα, as there are differences in its reversibility following fulvestrant withdrawal. ERα gene expression was not restored in MFR cells, and alternative intracellular phosphorylation signals were activated. By contrast, TFR cells exhibited plasticity of ERα gene expression and ERα-dependent growth; moreover, these cells were resensitized to estrogen and anti-estrogens. The difference in epigenetic regulation among individual cells might explain the difference in the plasticity of ERα expression. We also identified an MFR cell-activating HER/Src-Akt/MAPK pathway; thus, the specific inhibitors effectively blocked MFR cell growth. This finding implies the presence of multiple fulvestrant resistance mechanisms and suggests that the optimal therapies differ among individual tumors as a result of differing epigenetic mechanisms regulating ERα gene expression.

Activation Pathway of a Nucleoside Analog Inhibiting Respiratory Syncytial Virus Polymerase.

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA2α to Membrane.

The group IVA phospholipase A2, commonly called cytosolic phospholipase A2α (cPLA2α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cyclooxygenase and lipoxygenase pathways for production of prostaglandins and leukotrienes that regulate normal physiological processes and contribute to disease pathogenesis. cPLA2α is composed of an N-terminal C2 domain and a C-terminal catalytic domain that contains the Ser-Asp catalytic dyad. The catalytic domain contains phosphorylation sites and basic residues that regulate the catalytic activity of cPLA2α. In response to cell stimulation, cPLA2α is rapidly activated by posttranslational mechanisms including increases in intracellular calcium and phosphorylation by mitogen-activated protein kinases. In resting cells, cPLA2α is localized in the cytosol but translocates to membrane including the Golgi, endoplasmic reticulum, and the peri-nuclear membrane in response to increases in intracellular calcium. Calcium binds to the C2 domain, which promotes the interaction of cPLA2α with membrane through hydrophobic interactions. In this chapter, we describe assays used to study the calcium-dependent translocation of cPLA2α to membrane, a regulatory step necessary for access to phospholipid and release of arachidonic acid.

Dichloroacetate induces regulatory T-cell differentiation and suppresses Th17-cell differentiation by pyruvate dehydrogenase kinase-independent mechanism.

Recently, there has been a growing interest in the mechanism of action of dichloroacetate (DCA) for T-cell differentiation; however, this mechanism has not been elucidated in detail. Therefore, this study aimed to investigate the mechanism of action of DCA for Treg and Th17 differentiation with pyruvate dehydrogenase kinase (PDHK) inhibitor (AZD7545) and PDHK knockdown. Inhibitory activity of DCA and AZD7545 against recombinant PDHK and intracellular PDH phosphorylation was measured. The effects of DCA and AZD7545 on T-cell differentiation were assessed by analysing Foxp3+ T-cell populations for Treg differentiation and IL-17A production for Th17 differentiation. For reactive oxygen species (ROS) production, DCFDA was used as an indicator. Dichloroacetate and AZD7545 inhibited PDHK activity of recombinant PDHK and intracellular PDH phosphorylation. DCA was capable of inducing Treg differentiation and suppressing Th17 differentiation. The effects of DCA were independent of PDHK because neither AZD7545 nor knockdown of PDHK1 or PDHK3 affected T-cell differentiation. DCA was determined to be capable of inducing ROS production, and the effects of DCA on T-cell differentiation were shown to be dependent on ROS production. Dichloroacetate possesses Treg induction and Th17 suppression, which is independent of PDHK and dependent on ROS production.

Reelin Expression in Creutzfeldt-Jakob Disease and Experimental Models of Transmissible Spongiform Encephalopathies.

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1(Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration.

The M3 Phosphorylation Site Is Required for Trafficking and Biological Roles of PIN-FORMED1, 2, and 7 in Arabidopsis.

Asymmetrically localized PIN-FORMED (PIN) auxin efflux carriers play key roles in regulating directional intercellular auxin movement, generating local auxin gradients, and diverse auxin-mediated growth and development. The polar localization of PINs is controlled by phosphorylation in the central hydrophilic loop (HL) of PINs. Although the M3 phosphorylation site, including phosphorylatable 5 Ser/Thr residues, is conserved among long HL-PINs, its native role has only been characterized in PIN3. In this study, we examined the role of M3 phosphorylation site of PIN1, PIN2, and PIN7 in intracellular trafficking, phosphorylation, and biological functions of those PINs in their native expressing tissues. Phosphorylation-defective mutations of the phosphorylatable residues in the M3 site of PIN1-HL led to alteration in subcellular polarity of PIN1 and caused defects in PIN1-mediated biological functions such as cotyledon development, phyllotaxy of vegetative leaves, and development of reproductive organs. The M3 mutations of PIN7 interfered with its polar recycling in the root columella cell in response to gravity stimulus and partially disrupted root gravitropism. On the other hand, the M3 site of PIN2 was shown to be necessary for its targeting to the plasma membrane. In vitro phosphorylation assay showed that the M3 phosphorylation residues of PIN1 are the partial targets by PINOID kinase. Our data suggest that the M3 phosphorylation site is functionally conserved among long HL-PINs by playing roles for their subcellular trafficking and auxin-mediated developmental processes.

Abstract PR031

Background & Objectives: The physiological mechanisms that regulate tissue blood flow sufficiently are involved in the control of endothelium-dependent vasodilation. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), acts as an endothelium-dependent vasodilator. eNOS activity is regulated by several mechanisms including intracellular calcium concentration ([Ca2+]i) and phosphorylation of eNOS. Phosphorylation of Ser1177 increases Ca2+ sensitivity of the enzyme (1). Endothelium-dependent vasodilation is impaired by sevoflurane (2). The interaction of desflurane locally with the action of endothelial cells remains unknown. This study was carried out to elucidate the effects of desflurane and sevoflurane on endothelium-dependent vasodilation and the mechanisms of regulation of eNOS activity. Materials & Methods: With institutional approval, a rat descending aorta with the endothelium was suspended in a temperature-controlled organ bath for measurement of isometric tension. Changes in tension in response to phenylephrine (3×10–7 M) followed by acetylcholine (10–6 M) were recorded in the presence or absence of desflurane (5.7%, 11.4%; 1, 2 MAC for normal rats) or sevoflurane (2.4%, 4.8%; 1, 2 MAC for normal rats) to test endothelium-dependent vasodilation. Cultured bovine aortic endothelial cells were treated with bradykinin (10–6 M) in the presence or absence of desflurane or sevoflurane (11.4% and 4.8%, respectively). After treatments, Western blotting was used to determine eNOS Ser1177 phosphorylation, and measurement of the fluorescence intensity of Fluo-3/AM, as a [Ca2+]i indicator, was used to study the changes of [Ca2+]i. Statistical analysis was performed using Kruskal-Wallis’ H-test followed by Newman-Keuls test for multiple comparisons. A P value < 0.05 was considered statistically significant. Results: Endothelium-dependent vasodilation of the rat aorta and production of NO was inhibited by both anesthetics. The inhibitory effect of desflurane was greater than that of sevoflurane (Figure 1A, 1B). Western blotting revealed that phosphorylation of eNOS Ser1177 was inhibited by sevoflurane but not by desflurane (Figure 1C). Live cell calcium imaging revealed that bradykinin-induced increase in [Ca2+]i was suppressed by desflurane but not by sevoflurane (Figure 1D).Conclusion: The findings suggest that sevoflurane inhibits endothelium-dependent vasodilation caused by inhibition of eNOS Ser1177 phosphorylation, whereas desflurane inhibits vasodilation by inhibition of calcium signal transduction of eNOS activity. This difference in inhibitory mechanisms might reflect the different degrees of inhibitory effects of sevoflurane and desflurane on endothelium-dependent vasodilation.