Plasma Membrane Fraction Research Articles

Identification of novel therapeutic targets for histone 3 mutated children’s brain tumour, using unique tumour cell surface proteomic signatures

Abstract Aims Improvements in the treatments for childhood and adolescent brain tumours, High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), have not advanced much and they continue to carry a very poor prognosis. These brain tumours are now defined by mutations affecting histone 3 proteins, indeed 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. We hypothesized that the histone 3 mutant tumours will have distinct mutation specific surfactome (cell membrane proteins) signature. Method We therefore analysed the cell surface proteomics of pHGG and DIPG, in order to identify novel targets for therapy. We have at first isolated the cell membrane fractions from a range of patient cells carrying different histone 3 mutations (G34R, G34V), relative to wild type histone 3. A comparative quantitative mass-spectrometry analyses of these cell surface membrane fractions is then performed. Results The results obtained to date demonstrated unique differential cell membrane expression patterns which correlated to specific mutation type. For example, increased expression of Ras-related proteins Rab-3, Rab-3D is detected only in histone H3.3-G34R mutated cell line in comparison. Conclusion Identification and analyses of these unique cell membrane proteins’ association with specific in H3.3 mutation in pHGG, will help to identify precise mutation specific therapeutic targets, benefiting the patients to receive therapy based on tumour’s molecular signature.

Curium(III) speciation in the presence of microbial cell wall components

Trivalent actinides such as Cm(III) are able to strongly interact with microbes and especially with bacterial cell walls. However, detailed knowledge of the influence of different cell wall components is somewhat lacking. For this investigation, we studied the formation of aqueous Cm(III) complexes with cell wall components (e.g., lipopolysaccharide, peptidoglycan, and plasma membranes) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). For all systems, two specific Cm(III) complexes with the biomacromolecules were observed as a function of pH. Specifically, Cm(III) was found to bind to phosphate and carboxyl groups present in the structure of the biomacromolecules. Stability constants and luminescence parameters of the specific Cm(III) complexes were determined and are presented. The pH of the surrounding aqueous solution, the plasma membrane concentration, and proteins included in the crude plasma membrane fraction were found to significantly impact the complexation of Cm(III). The Cm(III) luminescence spectra with plasma membranes, cell wall polymers, as well as Gram-negative (Sporomusa sp. MT-2.99 and Pseudomonas fluorescens) and Gram-positive (Paenibacillus sp. MT-2.2) bacteria will be explained by linear combination fitting using the investigated components.

RabA2b Overexpression Alters the Plasma-Membrane Proteome and Improves Drought Tolerance in Arabidopsis.

Rab proteins are small GTPases that are important in the regulation of vesicle trafficking. Through data mining, we identified RabA2b to be stress responsive, though little is known about the involvement of RabA in plant responses to abiotic stresses. Analysis of the RabA2b native promoter showed strong activity during osmotic stress, which required the stress hormone Abscisic acid (ABA) and was restricted to the vasculature. Sequence analysis of the promoter region identified predicted binding motifs for several ABA-responsive transcription factors. We cloned RabA2b and overexpressed it in Arabidopsis. The resulting transgenic plants were strikingly drought resistant. The reduced water loss observed in detached leaves of the transgenic plants could not be explained by stomatal aperture or density, which was similar in all the genotypes. Subcellular localization studies detected strong colocalization between RabA2b and the plasma membrane (PM) marker PIP2. Further studies of the PM showed, for the first time, a distinguished alteration in the PM proteome as a result of RabA2b overexpression. Proteomic analysis of isolated PM fractions showed enrichment of stress-coping proteins as well as cell wall/cuticle modifiers in the transgenic lines. Finally, the cuticle permeability of transgenic leaves was significantly reduced compared to the wild type, suggesting that it plays a role in its drought resistant properties. Overall, these data provide new insights into the roles and modes of action of RabA2b during water stresses, and indicate that increased RabA2b mediated PM trafficking can affect the PM proteome and increase drought tolerance.

Peptidyl-prolyl cis-trans isomerase A participates in the selenium transport into the rat brain.

Selenium, an essential micronutrient, plays vital roles in the brain. Selenoprotein P (SELENOP), a major plasma selenoprotein, is thought to transport selenium to the brain. However, Selenop-knockout mice fed a diet containing an adequate amount of selenium shows no objective neurological dysfunction which is observed in the selenium-deficient diet-fed Selenop-knockout mice. This fact indicated that selenium from low-mass selenium-source compounds can be transported by SELENOP-independent alternative pathways to the brain. In this study, to obtain the basic information about the SELENOP-independent transport pathways, we performed ex vivo experiments in which the rat brain cell membrane fraction was analyzed to find selenium-binding and/or -interactive proteins using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several membrane proteins with the cysteine (C) thiol were found to be reactive with STS through the thiol-exchange reaction. One of the C-containing proteins in the brain cell membrane fraction was identified as peptidyl-prolyl cis-trans isomerase (PPIase) A from tryptic fragmentation experiments and database search. Among the 4 C residues in rat PPIase A, 21st C was proved to react with STS by assessment using C mutated recombinant proteins. PPIase A is ubiquitously expressed and also associates with a variety of biologically important events such as immunomodulation, intracellular signaling, transcriptional regulation and protein trafficking. Consequently, PPIase A was thought to participate in the selenium transport into the rat brain.

Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT.

Modification of the lipid A portion of LPS with cationic monosaccharides provides resistance to polymyxins, which are often employed as a last resort to treat multidrug-resistant bacterial infections. Here, we describe the use of fluorescent polyisoprenoids, liquid chromatography-mass spectrometry, and bacterial genetics to probe the activity of membrane-localized proteins that utilize the 55-carbon lipid carrier bactoprenyl phosphate (BP). We have discovered that a substantial background reaction occurs when B-strain E. coli cell membrane fractions are supplemented with exogenous BP. This reaction involves proteins associated with the arn operon, which is necessary for the covalent modification of lipid A with the cationic 4-aminoarabinose (Ara4N). Using a series of arn operon gene deletion mutants, we identified that the modification was dependent on ArnC, which is responsible for forming BP-linked Ara4N, or ArnT, which transfers Ara4N to lipid A. Surprisingly, we found that the majority of the Ara4N-modified isoprenoid was due to the reverse reaction catalyzed by ArnT and demonstrate this using heat-inactivated membrane fractions, isolated lipopolysaccharide fractions, and analyses of a purified ArnT. This work provides methods that will facilitate thorough and rapid investigation of bacterial outer membrane remodeling and the evaluation of polyisoprenoid precursors required for covalent glycan modifications.

Distinct Effects of Escitalopram and Vortioxetine on Astroglial L-Glutamate Release Associated with Connexin43

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.

Characterization of the O-Glycoproteome of Tannerella forsythia.

ABSTRACTTannerella forsythia is a Gram-negative oral pathogen known to possess an O-glycosylation system responsible for targeting multiple proteins associated with virulence at the three-residue motif (D)(S/T)(A/I/L/V/M/T). Multiple proteins have been identified to be decorated with a decasaccharide glycan composed of a poorly defined core plus a partially characterized species-specific section. To date, glycosylation studies have focused mainly on the two S-layer glycoproteins, TfsA and TfsB, so the true extent of glycosylation within this species has not been fully explored. In the present study, we characterize the glycoproteome of T. forsythia by employing FAIMS-based glycopeptide enrichment of a cell membrane fraction. We demonstrate that at least 13 glycans are utilized within the T. forsythia glycoproteome, varying with respect to the presence of the three terminal sugars and the presence of fucose and digitoxose residues at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides, we utilized trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. Reducing the chemical complexity of glycopeptides dramatically improved the number of glycopeptides identified and our ability to localize glycosylation sites by ETD fragmentation, leading to the identification of 312 putative glycosylation sites in 145 glycoproteins. Glycosylation site analysis revealed that glycosylation occurs on a much broader motif than initially reported, with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The prevalence of this broader glycosylation motif in the genome suggests the existence of hundreds of potential O-glycoproteins in this organism.IMPORTANCE Tannerella forsythia is an oral pathogen associated with severe forms of periodontal disease characterized by destruction of the tooth’s supporting tissues, including the bone. The bacterium releases a variety of proteins associated with virulence on the surface of outer membrane vesicles. There is evidence that these proteins are modified by glycosylation, and this modification is essential for virulence in producing disease. We have utilized novel techniques coupled with mass spectrometry to identify over 13 glycans and 312 putative glycosylation sites in 145 glycoproteins within T. forsythia. Glycosylation site analysis revealed that this modification occurs on a much broader motif than initially reported such that there is a high prevalence of potential glycoproteins in this organism that may help to explain its role in periodontal disease.

MP66-04 THE ROLE OF CHROMATIN AND HISTONE SUCCINYLATION AS A POST-TRANSLATIONAL MODIFICATION IN NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-04 THE ROLE OF CHROMATIN AND HISTONE SUCCINYLATION AS A POST-TRANSLATIONAL MODIFICATION IN NON-MUSCLE INVASIVE BLADDER CANCER Genesis Rivera-Marquez, Beatriz Walter, Maria Merino, and Vladimir Valera Genesis Rivera-MarquezGenesis Rivera-Marquez More articles by this author , Beatriz WalterBeatriz Walter More articles by this author , Maria MerinoMaria Merino More articles by this author , and Vladimir ValeraVladimir Valera More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder cancer has been characterized as one of the most genomically unstable cancers. Epigenetic events such as DNA methylation and histone acetylation have been described to play key roles in the disease onset and progression. These events are controlled ultimately by post-translational modifications (PTM) of proteins. In this study, we aimed to characterize a recently described PTM such as protein succinylation and its role in non-muscle invasive bladder cancer pathogenesis. METHODS: Well-characterized cell lines representing non-muscle invasive bladder cancer including MGH-U3 (pTa/pT1-derived), T24 (pTa-derived), RT4 (pT1-derived), RT-112 (pTa) were analyzed. Additionally, two muscle-invasive cancer derived cell lines UM-UC3 and SW-780 were included for comparison. All analysis were normalized to the reference Primary Bladder Epithelial cells (untransformed). Cell compartment fractionation was achieved by centrifugation under detergent, and included cell membrane, cytoplasmic, nuclear soluble and chromatin fractions. Extracts were then probed with Anti-Pan-Succinyl Lysine antibody. Histone-3 Acetylation and methylation were also tested. Chromatin immunoprecipitation with pan-succinyl-lysine was then performed and genomic DNA sent for Next Generation Sequencing (Chip-Seq) to identify transcription factors bound to succinylated chromatin. RESULTS: All cell compartments contained succinylated proteins, predominantly the cytosolic and chromatin/histone fractions. Histone-3 succinylation was prominent in all cancer cell lines predominantly the NMIBC ones. However, there was no clear association between succinylation and p53 and/or FGFR3 mutation status. Chip-seq analysis demonstrated that most of the represented marks on sequencing mapped to intergenic regions (>50%) and were associated with euchromatin markers, i.e, associated with gene activation. Metabolomic profiling of the cells also varied, ranging from mostly glycolytic pattern to predominatly ox-phos dependent. Accumulation of TCA-intermediaries such as Acetyl-Co-A, Fumarate, Citrate, and Succinate was marked in the NMIBC cells. CONCLUSIONS: Our results suggests that succinylation of chromatin components may be important for regulating nuclear functions, including chromatin dynamics and transcription and therefore in the pathogenesis of non-muscle invasive bladder cancer. Source of Funding: NIH Intramural Research Program © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1130-e1130 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Genesis Rivera-Marquez More articles by this author Beatriz Walter More articles by this author Maria Merino More articles by this author Vladimir Valera More articles by this author Expand All Advertisement Loading ...

Some Kinetic Features of Na,K-ATPase and Sensitivity to Noradrenaline

A comparative kinetic analysis of albino rat brain synaptic and kidney plasma membrane fraction Na,K-ATPase was performed to comprehend the different levels of sensitivity of these fractions to the neurotransmitter noradrenaline. Noradrenaline (NA) inhibits the rat brain synaptic membrane Na,K-ATPase, changes the stoichiometry of Na+ and K+ and shifts the enzyme system from an MgATP to an Mg2+ dependent cycle. While the kidney plasma membrane fraction Na,K-ATPase is not sensitive to noradrenaline. To investigate the mechanism underlying this difference, we studied enzyme velocity dependence on the concentration of Mg2+. The 1/V = f(Mg2+) function has shown different kinetic features for the synaptic and kidney plasma membrane Na,K-ATPase. With the addition of ethylene glycol tetraacetic acid (EGTA) to the reaction medium the geometric form of 1/V = f(Mg2+) function is affected differently. We thereafter measured the essential activator number for Na+ and K+ with, in excess Mg2+. The results of these experiments reveal that, contrary to the synaptic membrane Na,K-ATPase, the kidney plasma membrane fraction Na,K-ATPase does not possess an Mg2+ dependent cycle and noradrenaline exhibits different modulatory effects on the enzyme system.

Standardization and validation of assay of selected omega-3 and omega-6 fatty acids from phospholipid fraction of red cell membrane using gas chromatography with flame ionization detector

BackgroundDocosahexaenoic acid (DHA) is an important structural component of human brain and retina. Evidence exists linking nutritional status of pregnant mothers and cognitive functions of their born infants. The DHANI (Maternal DHA Supplementation and Offspring Neurodevelopment in India) trial was implemented to evaluate the effect of maternal supplementation with DHA during pregnancy and for 6 months following delivery on motor and mental development of infants at 1 and 12 months. We describe here the standardization and validation of an assay for measurement of selected omega-3 and omega-6 fatty acids from the phospholipid fraction of red blood cells to assess their status in mothers at baseline, delivery and 6 months post-delivery and for infants in cord blood and at 1 and 12 months of age. The validated method has been used for the analysis of samples for DHANI.MethodsLipids were extracted from a pool of red blood cells, separated using thin layer chromatography. The phospholipid fraction was esterified, and fatty acids were separated by gas chromatography using a flame ionization detector.ResultThe method accuracy for DHA was between 97 - 98% and between 91 - 95% for arachidonic acid (AA) at three different concentrations. The intra-assay and inter-assay coefficient of variation for the fatty acids ranged from 1.19 to 5.7% and 0.78 to 13.0% respectively. Intraclass correlation (ICC), as a measure of reproducibility, ranged between 0.689 and 0.996. A good linearity was observed for all the fatty acids between concentrations of 0.2–4 μg/ml.ConclusionThe standardized and validated method is suitable for implementation in large epidemiological studies for evaluation of fatty acids and in nutritional trials for assessment of fatty acid content of various lipid classes.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

High glucose induces trafficking of prorenin receptor and stimulates profibrotic factors in the collecting duct

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague–Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

A Novel Mechanism of Regulation for Exosome Secretion in the Diabetic Kidney.

Exosomes are nano-sized membrane vesicles generated by inward invagination of the endosomal membrane and released by the fusion of multivesicular bodies (MVB) with plasma membrane in various cell types (Fig. 1) (1). Once released, exosomes have diverse activities such as transmitting signals and biomolecules to neighboring or distant recipient cells and regulating the extracellular environment (2). Secreted exosomes have been shown to mediate intercellular communication among kidney cells and may play a key role in the pathogenesis of diabetic kidney disease (DKD) (3,4). Thus, it is important to understand the role and mechanisms of exosome secretion in the diabetic kidneys. Figure 1 Schematic representation of exosome biogenesis, transport, and secretion. Briefly, the exosome generation consists of three steps: biogenesis, intracellular transport, and release. A : Biogenesis. An inward invagination of the plasma membrane or membrane fractions from the trans -Golgi network (TGN) form early endosomes, which later mature into late endosomes/MVBs via inward budding of the endosomal membrane as intraluminal vesicles (conventional mediators include endosome sorting complex required for transport [ESCRT], ceramide/neutral sphingomyelinase 2 [nSMase2], tetraspanins, ALG-2 interacting protein X [ALIX], etc.). B : MVB trafficking. The fate of MVBs may vary and depend on molecules and effectors mediated (e.g., RAB27A/B fuses with the plasma membrane, RAB7/11 fuses to lysosome/autophagosome, etc.). C : Exosome release. The membrane of MVB fuses with the plasma membrane, which …

α2A- and α2C-adrenoceptor expression and functionality in postmortem prefrontal cortex of schizophrenia subjects

Previous evidence suggests that α2-adrenoceptors (α2-AR) may be involved in the pathophysiology of schizophrenia. However, postmortem brain studies on α2-AR expression and functionality in schizophrenia are scarce. The aim of our work was to evaluate α2A-AR and α2C-AR expression in different subcellular fractions of prefrontal cortex postmortem tissue from antipsychotic-free (absence of antipsychotics in blood at the time of death) (n = 12) and antipsychotic-treated (n = 12) subjects with schizophrenia, and matched controls (n = 24). Functional coupling of α2-AR to Gα proteins induced by the agonist UK14304 was also tested. Additionally, Gα protein expression was also evaluated. In antipsychotic-free schizophrenia subjects, α2A-AR and α2C-AR protein expression was similar to controls in all the subcellular fractions. Conversely, in antipsychotic-treated schizophrenia subjects, increased α2A-AR expression was found in synaptosomal plasma membrane and postsynaptic density (PSD) fractions (+60% and +79% vs controls, respectively) with no significant changes in α2C-AR. [35S]GTPγS SPA experiments showed a significant lower stimulation of Gαi2 and Gαi3 proteins by UK14304 in antipsychotic-treated schizophrenia subjects, whereas stimulation in antipsychotic-free schizophrenia subjects remained unchanged. Gαo protein stimulation was significantly decreased in both antipsychotic-free and antipsychotic-treated schizophrenia subjects compared to controls. Expression of Gαi3 protein did not differ between groups, whereas Gαi2 levels were increased in PSD of schizophrenia subjects, both antipsychotic-free and antipsychotic-treated. Gαo protein expression was increased in PSD of antipsychotic-treated subjects and in the presynaptic fraction of antipsychotic-free schizophrenia subjects. The present results suggest that antipsychotic treatment is able to modify in opposite directions both the protein expression and the functionality of α2A-AR in the cortex of schizophrenia patients.

1147-P: Pseudo Insulin Resistance: Palmitate Inactivates Insulin Signaling but Stimulates Basal Glucose Uptake in 3T3-L1 Adipocytes

Increased levels of circulating free fatty acids (FFA) is the major risk factor for type 2 diabetes in obesity. While the long-term effects of increased FFA on adipose tissue likely involve latent inflammation, the short-term effects on insulin-sensitive adipocytes are less clear. We treated fully differentiated 3T3-L1 adipocytes with increased concentrations (0.3-1 mM) of a typical FFA, palmitate, for 2 days, and measured insulin stimulation of 2-deoxyglucose (2-DG) uptake. The insulin cascade and AMPK activities, Glut-1 and Glut-4 levels were measured by western blots (WB). Glut-4 translocation was measured either by WB after cell membrane fractionation, or by fluorescence analysis after lentiviral transduction of adipocytes with Glut-4 construct containing C-terminal mCherry and outer Myc epitope insertion in the Glut-4 extracellular loop. While fluorescence of mCherry reported the total Glut-4, that of anti-Myc antibody revealed Glut-4 exposed on the plasma membrane. Palmitate dose dependently increased basal 2-DG uptake, whereas insulin-stimulated uptake and Glut-4 translocation to the plasma membrane were significantly reduced only by high (1 mM) palmitate. High palmitate did not alter Glut-1 and Glut-4 expression, but reduced IRS phosphorylation (Tyr612) and completely ceased that of Akt (Thr308/Ser473) and AS160 (Ser318) in the absence or presence of insulin, as well as phosphorylation of ACC (Ser-79) by AMPK. These results suggest that the short-term effects of palmitate on adipocytes are associated with “pseudo” insulin resistance, i.e., an increased basal glucose uptake likely due to the “pull” mechanism for increased triglyceride turnover, and the loss of insulin stimulation possibly due to fill-up of fat depots. Disclosure N. Podkuychenko: None. S. Michurina: None. I. Stafeev: None. A. Y. Khapchaev: None. V. P. Shirinsky: None. A. V. Vorotnikov: None. Funding Russian Foundation for Basic Research (20-015-00471)

Aspartame induces cancer stem cell enrichment through p21, NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells

This study aimed to investigate the molecular effects of the common natural sugar glucose and artificial sweetener aspartame on cancer stem cell (CSC) population and cancer aggressiveness of PANC-1 human pancreas adenocarcinoma cells. According to our findings while aspartame exposure significantly increased the CSC population, high glucose had no effect on it. The epithelial-mesenchymal transition marker N-cadherin increased only in the aspartame group. The findings indicate that a high level of glucose exposure does not effect the invasion and migration of PANC-1 cells, while aspartame increases both of these aggressiveness criteria. The findings also suggest that a high concentration of glucose maintains CSC population through induction of nuclear Oct3/4 and differentiation to parental cells via increasing cytoplasmic c-myc. Aspartame exposure to PANC-1 cells activated AKT and deactivated GSK3β by increasing levels of ROS and cytoplasmic Ca+2, respectively, through T1R2/T1R3 stimulation. Then p-GSK3β(Ser9) boosted the CSC population by increasing pluripotency factors Oct3/4 and c-myc via NICD, GLI1 and p21. In the aspartame group, T1R1 silencing further increased the CSC population but decreased cell viability and suppressed the p21, NICD and GLI activation. The presence and amount of T1R subunits in the membrane fraction of PANC-1 cells are demonstrated for the first time in this study, as is the regulatory effect of T1R1's on CSC population. In conclusion, the present study demonstrated that long-term aspartame exposure increases CSC population and tumor cell aggressiveness through p21, NICD, GLI1. Moreover, while aspartame had no tumorigenic effect, it could potentially advance an existing tumor.

Direct Bioelectrocatalytic Oxidation of Glucose by Gluconobacter oxydans Membrane Fractions in PEDOT:PSS/TEG-Modified Biosensors.

Recent years have witnessed an ever-increasing interest in developing electrochemical biosensors based on direct electron transfer-type bioelectrocatalysis. This work investigates the bioelectrocatalytic oxidation of glucose by membrane fractions of Gluconobacter oxydans cells on screen-printed electrodes modified with thermally expanded graphite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Electrooxidation of glucose was shown to occur without the presence of electron transport mediators. Chronoamperometric and cyclic voltametric characteristics showed an increase of anodic currents at electrode potentials of 0–500 mV relative to the reference electrode (Ag/AgCl). The direct electron transfer effect was observed for non-modified PEDOT:PSS as well as for PEDOT:PSS linked with crosslinkers and conductive fillers such as polyethylene glycol diglycidyl or dimethyl sulfoxide. Bioelectrodes with this composite can be successfully used in fast reagent-free glucose biosensors.

Rapid Increase of Gastrointestinal P-Glycoprotein Functional Activity in Response to Etoposide Stimulation.

We previously reported that exposure of human colon adenocarcinoma (Caco-2) cells to the bitter substance phenylthiocarbamide (PTC) rapidly enhanced the transport function of P-glycoprotein (P-gp). In this study, we investigated the short-term effect of etoposide, another bitter-tasting P-gp substrate, on P-gp transport function in the same cell line. We found that etoposide exposure significantly increased both the P-gp protein level in the plasma membrane fraction and the efflux rate of rhodamine123 (Rho123) in Caco-2 cells within 10 min. The efflux ratio (ratio of the apparent permeability coefficient in the basal-to-apical direction to that in the apical-to-basal direction) of Rho123 in etoposide-treated cells was also significantly increased compared with the control. These results indicated that etoposide rapidly enhances P-gp function in Caco-2 cells. In contrast, P-gp expression in whole cells at both the mRNA and protein level was unchanged by etoposide exposure, compared with the levels in non-treated cells. Furthermore, etoposide increased the level of phosphorylated ezrin, radixin and moesin (P-ERM) proteins in the plasma membrane fraction of Caco-2 cells within 10 min. P-gp functional changes were blocked by YM022, an inhibitor of cholecystokinin (CCK) receptor. These results suggest that etoposide induces release of CCK, causing activation of the CCK receptor followed by phosphorylation of ERM proteins, which recruit intracellular P-gp for trafficking to the gastrointestinal membrane, thereby increasing the functional activity of P-gp.

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H2 O2 or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p<.05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p<.05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.