Crude Membrane Research Articles

Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain*

Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus.

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B.

Aim:Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR.Methods:Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B.Results:Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 μmol/L. Br-IQ17B is selective over other subtypes such as α4β2 and α3β4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [3H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive.Conclusion:We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.

Docosahexaenoic acid inhibits the invasion of MDA-MB-231 breast cancer cells through upregulation of cytokeratin-1.

Docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family, has been shown to reduce the invasion of the triple-negative breast cancer cell line MDA-MB-231, but the mechanism involved remains unclear. In the present study, a proteomic approach was used to define changes in protein expression induced by DHA. Proteins from crude membrane preparations of MDA-MB-231 cells treated with 100 µM DHA were separated by two-dimensional electrophoresis (2-DE) and differentially expressed proteins were identified using MALDI-TOF mass spectrometry. The main changes observed were the upregulation of Keratin, type Ⅱ cytoskeletal 1 (KRT1), catalase and lamin-A/C. Immunocytochemistry analyses confirmed the increase in KRT1 induced by DHA. Furthermore, in vitro invasion assays showed that siRNA against KRT1 was able to reverse the DHA-induced inhibition of breast cancer cell invasion. In conclusion, KRT1 is involved in the anti-invasive activity of DHA in breast cancer cells.

The Photosystem II D1-K238E mutation enhances electrical current production using cyanobacterial thylakoid membranes in a bio-photoelectrochemical cell.

The conversion of solar energy (SEC) to storable chemical energy by photosynthesis has been performed by photosynthetic organisms, including oxygenic cyanobacteria for over 3 billion years. We have previously shown that crude thylakoid membranes from the cyanobacterium Synechocytis sp. PCC 6803 can reduce the electron transfer (ET) protein cytochrome c even in the presence of the PSII inhibitor DCMU. Mutation of lysine 238 of the Photosystem II D1 protein to glutamic acid increased the cytochrome reduction rates, indicating the possible position of this unknown ET pathway. In this contribution, we show that D1-K238E is rather unique, as other mutations to K238, or to other residues in the same vicinity, are not as successful in cytochrome c reduction. This observation indicates the sensitivity of ET reactions to minor changes. As the next step in obtaining useful SEC from biological material, we describe the use of crude Synechocystis membranes in a bio-photovoltaic cell containing an N-acetyl cysteine-modified gold electrode. We show the production of significant current for prolonged time durations, in the presence of DCMU. Surprisingly, the presence of cytochrome c was not found to be necessary for ET to the bio-voltaic cell.

Acute systemic insulin intolerance does not alter the response of the Akt/GSK-3 pathway to environmental hypoxia in human skeletal muscle.

To investigate how acute environmental hypoxia regulates blood glucose and downstream intramuscular insulin signaling after a meal in healthy humans. Fifteen subjects were exposed for 4h to normoxia (NOR) or to normobaric hypoxia (HYP, FiO2=0.11) in a randomized order 40min after consumption of a high glycemic meal. A muscle biopsy from m. vastus lateralis and a blood sample were taken before (T0), after 1h (T60) and 4h (T240) in NOR or HYP and blood glucose levels were measured before exposure and every 30min. In HYP, blood glucose was reduced 100min (110.1±5.4 in NOR vs 89.5±4.7mgdl(-1) in HYP) and 130min (98.7±3.8 in NOR vs 85.6±4.9mgdl(-1) in HYP) after completion of a meal, which resulted in an 83% lower AUC in HYP compared to NOR (p=0.006). This coincided with 40% lower GLUT4 protein in the cytosolic fraction (p=0.013) and a tendency to increase in the crude membrane fraction (p=0.070) in HYP compared to NOR. At T240, blood glucose concentration was similar between HYP and NOR, whereas plasma insulin as well as phosphorylation of muscle Akt and GSK-3 was ~2-fold higher in HYP compared to NOR (p<0.05). In contrast, Rac1 protein was less abundant in the membrane fraction in HYP compared to NOR (p=0.003), reflecting lower activation. Acute environmental hypoxia initially reduced blood glucose response to a meal, possibly via an increase in GLUT4 abundance at the sarcolemmal membrane. Later on, whole body insulin intolerance developed independently of defects in conventional insulin signaling in skeletal muscle.

Taurine Targets the GluN2b-Containing NMDA Receptor Subtype.

As an endogenous neuromodulator in the CNS, Taurine interacts with TAG (6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide)-sensitive and TAG-insensitive receptors. Taurine activates the former by opening a Cl− channel and the latter by inhibiting the glutamate NMDA receptor. We sought to resolve the site of taurine’s interaction with the NMDA receptor. We recorded evoked field potential in medial prefrontal cortical slices and compared its dose-dependent inhibition by Ro25-6981, a selective antagonist for the GluN1/GluN2B NMDA receptor sub-type, in the absence or presence of taurine. The result revealed that inhibition of evoked responses mediated by taurine overlapped with that by Ro25-6981, suggesting that taurine modulates NMDA receptor by acting on the NMDA GluN1/GluN2B receptor sub-type. Displacement of specific binding of [3H]spermidine and of [3H]taurine to crude frontal cortical membranes by spermine and spermidine showed that polyamines and taurine may interact at a common binding site, possibly localized at the GluN1 or GluN2B subunit. We also tested for long-term taurine actions on glutamate receptor subunits using western blot determination of NMDA and AMPA receptor subunits expression in synaptosomal membranes prepared from rat frontal cortex following chronic taurine treatment. Thirty daily i.p. injections of taurine (100 mg/kg) significantly increased expression of the NMDA GluN2B, but not GluN1, subunit and decreased expression of the AMPA GluR2 subunit. The up-regulation of the GluN2B subunit suggests its long-term interaction with taurine, and supports its being a major target for taurine action. Down-regulation of the AMPA GluR2 subunit is possibly correlated to an increased recruitment of the GluR2-subunit-lacking, calcium-permeable subtype of AMPA receptor.

Abstract 15813: Loss-of-Function Mutation in Toll-Like Receptor 4 Partially Protects Against Peripheral and Cardiac Insulin Resistance During a Long-Term High-Fat Diet

Diabetes mellitus is an epidemic disease characterized by alterations in glucose transport, which is tightly regulated by a family of specialized proteins called the glucose transporters (GLUTs). Although diabetic cardiomyopathy is a common complication in diabetic patients, its pathogenesis is still not well understood. Toll-like receptor (TLR) 4, which plays a central role in pathogen recognition by the innate immune system, may also play a critical role in linking inflammation and metabolic disease. We hypothesized that TLR4 activation triggers cardiac insulin resistance. We used mice with a loss-of function mutation in TLR4 (C3H/HeJ) and age-matched wild-type (WT, C57BL/6N) mice (n=8/group) to investigate how feeding a high-fat diet (HFD, 60% kcal from fat) for 16 weeks affected whole-body and cardiac glucose metabolism. After 16 weeks, WT mice fed a HFD were obese and developed hyperglycemia and insulin resistance compared to WT mice on a control diet (10% kcal from fat). The C3H/HeJ mice were partially protected against HFD-induced obesity and insulin resistance. In the heart, WT mice fed a HFD had a 30% decrease (P&lt;0.05) in GLUT4 protein content as measured by Western Blot of cardiac crude membrane protein extracts. In contrast, the loss-of-function point mutation in TLR4 partially rescued cardiac GLUT4 content in the face of a HFD. Interestingly, there was a 40% increase (P&lt;0.05) in the novel GLUT isoform, GLUT8, in the heart when mice of either genotype were fed a HFD. Additionally, GLUT4 protein content was negatively (P&lt;0.05) correlated with GLUT8 content in the myocardium, suggesting that GLUT8 may act as a compensatory mechanism in the face of HFD-induced GLUT4 downregulation. Phosphorylated Akt, a key protein of the insulin signaling pathway, was positively (P&lt;0.05) correlated with GLUT4 content, while the basal/inactive form was negatively correlated. In conclusion, these data suggest that activation of TLR4 activation during diabetes and obesity alters glucose transport by an Akt mechanism, and as such is a pathogenic factor during peripheral and cardiac insulin resistance. Overall, TLR4 appears to be a key modulator in the cross-talk between inflammatory and metabolic pathways, as well as a potential therapeutic target for diabetes.

Analysis of hyaluronan synthase activity.

Hyaluronan (HA) is a component of the extracellular matrix that is involved in many physiological and pathological processes. As HA modulates several functions (i.e., cell proliferation and migration, inflammation), its presence in the tissues can have positive or negative effects. HA synthases (HAS) are a family of three isoenzymes located on the plasma membrane that are responsible for the production of such polysaccharide and, therefore, their activity is critical to determine the accumulation of HA in tissues. Here, we describe a nonradioactive method to quantify the HAS enzymatic activity in crude cellular membrane preparation.

Effects of partial pressure of oxygen on the modulation of syncytiotrophoblast potassium channels by reactive oxygen species

s / Placenta 35 (2014) A1eA112 A97 mouse knockout models of impaired sulfonation of structural molecules important for foetal development. Conclusion: Our novel mouse model is providing valuable insight into the critical role of placental sulphate transporters in meeting foetal nutrient demands during gestation. P2.115-N. PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE Benjamin P. Luscher , Michael Fine , Camilla Marini , Benjamin Clemencon , Christiane Albrecht , Matthias A. Hediger , Daniel V. Surbek , Marc U. Baumann a,b Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland; c Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland Objectives: Materno-fetal transplacental transport is crucial for the fetal well-being. The altered expression of placental transport proteins under specific pathophysiological conditions may affect the intrauterine environment. Pre-eclampsia is often associated with high maternal uric acid serum levels. The regulation of the placental uric transport system and its transporter glucose transporter (GLUT)-9 are not fully understood yet. The aim of this study was to investigate the placental urate transport and to characterize its transporter GLUT9. Methods: In this study we used a transepithelial transport (Transwell®) model to assess uric acid transport activity. Electrophysiological techniques and radioactive ligand up-take assays were used to measure transport activity of GLUT9 expressed in Xenopus oocytes. Results: In the Transwell/ model uric acid is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min at the linear stage. We could successfully over-express GLUT9 using the Xenopus laevis oocytes expression system. Chloride modulates the urate transport system: interestingly replacing chloride with iodine resulted in a complete loss of urate transport activity. We determined the IC50 of iodine at 30uM concentration. In radioactiveup-takeexperiments iodinehadnoeffectonuric acid transport. Conclusions: In vitro the “materno-fetal” transport of uric acid is slow. This indicates that in vivo the child is protected from short-term fluctuations of maternal uric acid serum concentrations. The different results regarding iodine-mediated regulation of GLUT9 transport activity between electrophysiological and radioactive ligand uptake experiments may suggest that iodine does not directly inhibit uric acid transport, but changes the mode of up-take from an electrogenic to an electroneutral transport. GLUT9 is not an uric acid uniporter, there are more ions involved in the transport. This may allow regulating uric acid transport by the change from an active to a passive transport. P2.116. UPTAKE MECHANISM OF AN ESTRIOL PRECURSOR, 16a-HYDROXY DEHYDROEPIANDROSTERONE SULFATE, AT THE BASAL PLASMA MEMBRANE OF HUMAN TERM PLACENTA Masatoshi Tomi , Hiromi Eguchi , Tomohiro Nishimura , Tetsuo Maruyama , Emi Nakashima a Keio University Faculty of Pharmacy, Tokyo, Japan; Keio University School of Medicine, Tokyo, Japan Objectives: Estriol is mostly secreted from placental syncytiotrophoblasts during pregnancy. 16a-Hydroxy dehydroepiandrosterone sulfate (16a-OH DHEAS) is an estriol precursor, which is synthesized by fetal liver-specific CYP3A7 (16a-hydroxylase). To achieve placental estriol synthesis, syncytiotrophoblasts require organic anion transporter(s) for 16a-OH DHEAS uptake at the basal plasma membrane (BM) facing fetal circulation. The purpose of this study is to clarify the mechanism underlying 16a-OH DHEAS uptake at the BM of human placental syncytiotrophoblasts. Methods: The uptake of [3H]16a-OH DHEAS was characterized using basal-enriched plasma membrane vesicles (BMVs) derived from human term placenta. The expression of transporters in BMVs was determined by western blot analysis. Results:Proteinexpressionsof organic anion transporter (OAT)4 (SLC22A11) and organic anion transporting polypeptide 2B1 (SLCO2B1)were enriched in human placental BMVs compared with crude membrane fractions, which is consistent with immunohistochemical observations of the basal localization of these transporters in syncytiotrophoblasts. Uptake of [3H]16a-OH DHEAS by BMVs was sodiumand chloride-independent and was trans-stimulated by preloaded glutarate. The uptake of [3H]16a-OH DHEAS was significantly cis-inhibited byDHEAS, estrone sulfate, andbromosulfophthalein, but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, and cimetidine. These transport characteristics of [3H]16a-OHDHEAS are in good agreement with those in human OAT4-transfected COS7 cells. Conclusion: OAT4 expressed at the basal plasma membrane of the syncytiotrophoblast appears to play a predominant role in the placental uptake of 16a-OH DHEAS from the fetal circulation and contribute to estriol synthesis during pregnancy. P2.117. NANOPARTICLE INTERACTION WITH EARLY HUMAN PLACENTA: PLACENTAL BARRIER MATTERS? Liudmila Nikitina , Gottfried Dohr , Herbert Juch a,b a Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria; b Institute of Human Genetic, Medical University of Graz, Graz,

An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay (32)P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 G: . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

Diet effects on glucose absorption in the small intestine of neonatal calves: Importance of intestinal mucosal growth, lactase activity, and glucose transporters

Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters.

Recombinant outer membrane protein: a potential candidate for Th17 based vaccine against Klebsiella pneumoniae. (VAC7P.967)

Abstract Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae. Recently, the emergence of antibiotic resistant strains demands an effective vaccine against these bacteria. We have previously shown that intranasal immunization with heat-killed K. pneumoniae or crude outer membrane proteins isolated from K. pneumoniae induced antigen specific Th17 responses and these Th17 cells conferred serotype independent protection against various clinical isolates of K. pneumoniae including the recently described multidrug resistant New Delhi Metallo-beta-lactamase-1 strain. To develop a clinically relevant Klebsiella vaccine, we cloned single OMP genes by PCR from K. pneumoniae and successfully expressed one recombinant outer membrane protein, OmpX, in the BL21 E. coli strain. The purified recombinant OmpX was recognized by Klebsiella immune serum by direct ELISA and also recognized by Th17 cells from Klebsiella immunized mice. In vivo, intranasal immunization of the purified OmpX induced robust mucosal Th17 responses and left IL-17 producing gamma-delta T cells unaffected. Ongoing work will determine whether these Th17 responses will result in serotype independent protection against live bacterial challenge including the multidrug resistant strains.

Src‐kinase mediates angiotensin II‐induced potentiation in TRPV4 agonist‐evoked calcium transients in hypothalamic immortalized neuronal cell line 4B (860.25)

We have previously demonstrated that in bile duct ligated rats, an animal model of inappropriate vasopressin (AVP) release, TRPV4 protein expression and membrane trafficking is increased in AVP neurons. Here, we used an in vitro approach with an immortalized neuronal AVP expressing cell line (4B neurons) to investigate the possible regulation of TRPV4 by angiotensin II (Ang II). We characterized the presence of TRPV4 mRNA and protein in 4B cells. After Ang II (100nM;1 hr) treatment significantly increased TRPV4 levels in crude membrane fractions (p&lt;0.001) and tyrosine phosphorylation of TRPV4 (p&lt;0.001). Using calcium sensitive dye Fura‐2AM, we noted that Ang II treated cells exhibited increased calcium transients in response to TRPV4 agonist, GSK1016790A (20nM, p&lt;0.05). This increase was blocked by the Losartan (Ang II receptor antagonist) and Src‐kinase inhibitor, PP2, but not by its analog PP3. Our data indicate that Ang II may alter the phosphorylation status of TRPV4 through Src‐kinases.Grant Funding Source: Supported by: R01 HL62569

Characterization of the membrane proteome and N-glycoproteome in BV-2 mouse microglia by liquid chromatography-tandem mass spectrometry

BackgroundMicroglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited.ResultsTo generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses.ConclusionsHerein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases.

Distinct biochemical activities and heat shock responses of two UDP-glucose sterol glucosyltransferases in cotton

UDP-glucose sterol glucosyltransferase (SGT) are enzymes typically involved in the production of sterol glycosides (SG) in various organisms. However, the biological functions of SGTs in plants remain largely unknown. In the present study, we identified two full-length GhSGT genes in cotton and examined their distinct biochemical properties. Using UDP-[U-14C]-glucose and β-sitosterol or total crude membrane sterols as substrates, GhSGT1 and GhSGT2 recombinant proteins were detected with different enzymatic activities for SG production. The addition of Triton (X-100) strongly inhibited the activity of GhSGT1 but caused an eightfold increase in the activity of GhSGT2. The two GhSGTs showed distinct enzyme activities after the addition of NaCl, MgCl2, and ZnCl2, indicating that the two GhSGTs exhibited distinct biochemical properties under various conditions. Furthermore, after heat shock treatment, GhSGT1 showed rapidly enhanced gene expression in vivo and low enzyme activity in vitro, whereas GhSGT2 maintained extremely low gene expression levels and relatively high enzyme activity. Notably, the GhSGT2 gene was highly expressed in cotton fibers, and the biochemical properties of GhSGT2 were similar to those of GhCESA in favor for MgCl2 and non-reduction reaction condition. It suggested that GhSGT2 may have important functions in cellulose biosynthesis in cotton fibers, which must be tested in the transgenic plants in the future. Hence, the obtained data provided insights into the biological functions of two different GhSGTs in cotton and in other plants.

Protein expression of kidney and liver bilitranslocase in rats exposed to mercuric chloride—A potential tissular biomarker of toxicity

Bilitranslocase (BTL) is a plasma membrane carrier that transports organic anions of physiological and pharmacological interest. It is expressed in basolateral plasma membrane of kidney and liver. BTL has been recently described as a marker of transition from normal tissue to its neoplastic transformation in human kidney. Inorganic mercury is a major environmental contaminant that produces many toxic effects. Previous reports have described an interaction between BTL and mercuric ions. This study was designed to evaluate the renal and hepatic expression of BTL in rats exposed to a nephrotoxic and hepatotoxic dose of HgCl2. Male rats were treated with a single injection of HgCl2 at a dose of 4mg/kg body wt, i.p. (HgCl2 group). Control rats received the vehicle alone (Control group). Studies were carried out 18h after injection. Afterwards, the kidneys and livers were excised and processed for histopathological studies or immunoblot (homogenates and crude membranes) techniques. In rats treated with HgCl2, immunoblotting showed a significant decrease in the abundance of BTL in homogenates and plasma membranes from kidney and liver. BTL decrease of expression might reflect the grade of damage in renal tubule cells and in hepatocytes. Thus, BTL might be postulated as a new biomarker of tissue toxicity induced by mercury.

Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

Modafinil restores methamphetamine induced object-in-place memory deficits in rats independent of glutamate N-methyl-d-aspartate receptor expression

BackgroundChronic methamphetamine (meth) abuse in humans can lead to various cognitive deficits, including memory loss. We previously showed that chronic meth self-administration impairs memory for objects relative to their location and surrounding objects. Here, we demonstrate that the cognitive enhancer, modafinil, reversed this cognitive impairment independent of glutamate N-methyl-d-aspartate (GluN) receptor expression MethodsMale, Long-Evans rats underwent a noncontingent (Experiment 1) or contingent (Experiment 2) meth regimen. After one week of abstinence, rats were tested for object-in-place recognition memory. Half the rats received either vehicle or modafinil (100mg/kg) immediately after object familiarization. Rats (Experiment 2) were sacrificed immediately after the test and brain areas that comprise the key circuitry for object in place performance were manually dissected. Subsequently, glutamate receptor expression was measured from a crude membrane fraction using Western blot procedures. ResultsSaline-treated rats spent more time interacting with the objects in changed locations, while meth-treated rats distributed their time equally among all objects. Meth-treated rats that received modafinil showed a reversal in the deficit, whereby they spent more time exploring the objects in the new locations. GluN2B receptor subtype was decreased in the perirhinal cortex, yet remained unaffected in the prefrontal cortex and hippocampus of meth rats. This meth-induced down regulation occurred whether or not meth experienced rats received vehicle or modafinil. ConclusionsThese data support the use of modafinil for memory impairment in meth addiction. Further studies are needed to elucidate the neural mechanisms of modafinil reversal of cognitive impairments.

Possible association of Neu2 with plasma membrane fraction from mouse thymus exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU-Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP-40 or DOC/NP-40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris-buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane-rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2-transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.