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Purpose: To investigate the affinity of a bis-indole alkaloid - voacamine from Voacanga Africana Stapf for IL-2Rα - and its immunosuppressive effect on concanavalin A-induced T cell proliferation and lipopolysaccharide -induced B cell proliferation in vitro. Methods: Surface plasmon resonance imaging (SPRi) was used to screen the target protein of voacamine, while CCK-8 kit was used to evaluate cytotoxicity. Mitogen-induced proliferation assay was carried out to assess the inhibitory effect of voacamine on Con A-induced T cell proliferation and LPSinduced B cell proliferation. The binding characteristics of voacamine were investigated using a binding model with IL-2Rα constructed based on molecular docking simulation. Results: Voacamine had a high-affinity for IL-2Rα with an equilibrium dissociation constant (KD) of 1.85×10-8 M. Cytotoxicity data showed that voacamine did not exhibit cytotoxicity at concentrations lower than 0.32 µM. However, it exerted significant immunosuppressive effect on B cells at a lower concentration, but had no influence on proliferation of T cells. Autodock results indicate that voacamine has a good interaction with the enzyme active site. Conclusion: Voacamine and its analogues exert influence on the immune system.

Spatial confinement is a desirable successful strategy to trap sulfur within its porous host and has been widely applied in lithium–sulfur (Li–S) batteries. However, physical confinement alone is currently not enough to reduce the lithium polysulfide (Li2Sn, 4 ≤ n ≤ 8, LIPSs) shuttle effect with sluggish LIPS-dissolving kinetics. In this work, we have integrated spatial confinement with a polar catalyst, and designed a three-dimensional (3D) interconnected, Co decorated and N doped porous carbon nanofiber (Co/N-PCNF) network. This Co/N-PCNF film serves as a freestanding host for sulfur trapping, which could effectively facilitate the infiltration of electrolyte and electron transport. In addition, the polar Co species possess strong chemisorption with LIPSs, catalyzing their reaction kinetics as well. As a result of this rational design and integration, the Co/N-PCNF@S cathode with a sulfur loading of 2 mg cm−2 exhibits a high initial discharge capacity of 878 mA h g−1 at 1C, and maintains a discharge capacity of 728 mA h g−1 after 200 cycles. Even with high sulfur loading of 9.33 mg cm−2, the cathode still keeps a stable areal capacity of 7.16 mA h cm−2 at 0.2C after 100 cycles, which is much higher than the current areal capacity (4 mA h cm−2) of commercialized lithium-ion batteries (LIBs). This rational design may provide a new approach for future development of high-density Li–S batteries with high sulfur loading.

Sodium-ion batteries (SIBs) have recently drawn great interest as an alternative due to their lower cost compare to lithium-ion batteries (LIBs). As in the practical application in LIBs manufacturing, Cu and Al foils are generally used as the current collectors for the anode and cathode of SIBs respectively. The concern for amorphous Li-Al alloy formation at ∼0.2 V is the main reason for selecting a different metallic (i.e. Cu) current collector. However, it is not the case for the SIBs since Na-Al alloys are formed at much lower potentials. In this paper, we demonstrate that the electrochemistry of SIBs allows an Al current collector to be used for the SIB anode without any adverse effect. The cell performance using a high capacity Sb2S3-reduced graphene oxide (Sb2S3-rGO) composite anode is undistinguishable between the use of Cu and Al current collectors. The reversible storage of Na+ in this Sb2S3-rGO composite is evaluated on both Cu-foil and Al-foil current collectors and comparable results are obtained: high desodiation specific capacities of ∼555 mAh g−1 (on Al-foil current collector) and 537 mAh g−1 (on Cu-foil current collector) for 70 cycles at 100 mA g−1. Therefore, the Al current collector can replace Cu in SIBs, which simplifies the construction of SIB cells and reduces the material cost in SIB production.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler’s diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+concentration [Ca2+]i). The ST elevation of [Ca2+]iwas from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

WOS: 000444554700003, PubMed ID: 29796531, The relative stabilities of different chemical arrangements of Pd-Ir and Au-Rh nanoalloys (and their pure metal equivalents) are studied, for a range of compositions, for fcc truncated octahedral 38- and 79-atom nanoparticles (NPs). For the 38-atom NPs, comparisons are made of pure and alloy NPs supported on a TiO2(110) slab. The relative energies of different chemical arrangements are found to be similar for Pd-Ir and Au-Rh nanoalloys, and depend on the cohesive and surface energies of the component metals. For supported nanoalloys on TiO2, the interaction with the surface is greater for Ir (Rh) than Pd (Au): most of the pure NPs and nanoalloys preferentially bind to the TiO2 surface in an edge-on configuration. When Au-Rh nanoalloys are bound to the surface through Au, the surface binding strength is lower than for the pure Au NP, while the Pd-surface interaction is found to be greater for Pd-Ir nanoalloys than for the pure Pd NP. However, alloying leads to very little difference in Ir-surface and Rh-surface binding strength. Comparing the relative stabilities of the TiO2-supported NPs, the results for Pd-Ir and Au-Rh nanoalloys are the same: supported Janus NPs, whose Ir (Rh) atoms bind to the TiO2 surface, bind most strongly to the surface, becoming closer in energy to the core-shell configurations (Ir@Pd and Rh@Au) which are favoured for the free particles., Xiamen University; EPSRC Critical Mass Grant TOUCAN [EP/J010804/1]; EPSRC [EP/L000202]; TOUCAN grant; Royal Society International Exchange Grant [IE140712], T-EF is grateful for funding from the Graduate School of Xiamen University. RLJ and ID acknowledge support through EPSRC Critical Mass Grant (EP/J010804/1) TOUCAN. Calculations were performed on the following HPC facilities: The University of Birmingham Bluebear HPC facility (see http://www.bear.bham.ac.uk/bluebear for more details) and the UK's national HPC facility, ARCHER, both via membership of the UK's HPC Materials Chemistry Consortium, which is funded by EPSRC (EP/L000202), and via the TOUCAN grant. ZYL acknowledges Royal Society International Exchange Grant IE140712. LP acknowledges the French METSA network and Corinne Ulhaq (IPCMS, Strasbourg) for STEM imaging of Pd-Ir nanoparticles.

In this work, an electroanalytical platform for nereistoxin (NRT)-related insecticide detection is proposed on the basis of NRT induced DNA conformational switching and exonuclease III (Exo III) assisted target recycling. NRT-related insecticides were first hydrolyzed and converted into NRT with two thiol groups (–SH). Then, a cytosine–Ag+–cytosine (C–Ag+–C) mismatched base pair was adopted to induce a blunt-ended hairpin configuration of HP DNA. In the presence of converted NRT, it could take up Ag+ from HP DNA to change its conformation from a hairpin to single-stranded structure (HP ssDNA). Thereafter, the obtained HP ssDNA was further hybridized with an H1 hairpin probe on the electrode surface to trigger the Exo III cleavage process, releasing HP ssDNA for recycling leaving the G-quadruplex fragment of H1, which was used for hemin/G-quadruplex complex formation. The reversible redox reaction of Fe(III)/Fe(II) of hemin gave a remarkable electrochemical response for quantitative determination of the NRT-related insecticides. As an analytical model, a low detection limit of 3.9 ng L−1 and a wide linear range of 0.01–1500 μg L−1 with excellent selectivity were achieved for cartap detection. The proposed method also displayed great applicability for cartap detection in agricultural products.

The two major limitations in the application of SnO2 for lithium-ion battery (LIB) anodes are the large volume variations of SnO2 during repeated lithiation/delithiation processes and a large irreversible capacity loss during the first cycle, which can lead to a rapid capacity fade and unsatisfactory initial Coulombic efficiency (ICE). To overcome these limitations, we developed composites of ultrafine SnO2 nanoparticles and in situ formed Co(CoSn) nanocrystals embedded in an N-doped carbon matrix using a Co-based metal–organic framework (ZIF-67). The formed Co additives and structural advantages of the carbon-confined SnO2/Co nanocomposite effectively inhibited Sn coarsening in the lithiated SnO2 and mitigated its structural degradation while facilitating fast electronic transport and facile ionic diffusion. As a result, the electrodes demonstrated high ICE (82.2%), outstanding rate capability (~ 800 mAh g−1 at a high current density of 5 A g−1), and long-term cycling stability (~ 760 mAh g−1 after 400 cycles at a current density of 0.5 A g−1). This study will be helpful in developing high-performance Si (Sn)-based oxide, Sn/Sb-based sulfide, or selenide electrodes for LIBs. In addition, some metal organic frameworks similar to ZIF-67 can also be used as composite templates.

The structures and surface adsorption sites of Pd-Ir nanoalloys are crucial to the understanding of their catalytic performance because they can affect the activity and selectivity of nanocatalysts. In this article, density functional theory (DFT) calculations are performed on bare Pd-Ir nanoalloys to systematically explore their stability and chemical ordering properties, before studying the adsorption of CO on the nanoalloys. First, the structural stability of 38-atom and 79-atom truncated octahedral (TO) Pd-Ir nanoalloys are investigated. Then the adsorption properties and preferred adsorption sites of CO on 38-atom Pd-Ir nanoalloys are considered. The PdshellIrcore structure, which has the lowest energy of all the considered isomers, exhibits the highest structural stability, while the PdcoreIrshell configuration is the least stable. In addition, the adsorption strength of CO on Ir atoms is found to be greater than on Pd for Pd-Ir nanoclusters. The preferred adsorption sites of CO on pure Pd and Ir clusters are in agreement with calculations and experiments on extended Pd and Ir surfaces. In addition, d-band center and charge effects on CO adsorption strength on Pd-Ir nanoalloys are analyzed by comparison with pure clusters. The study provides a valuable theoretical insight into catalytically active Pd-Ir nanoalloys.

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na(+)/H(+) exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na(+)/H(+) exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.

The Na+/H+ exchanger 3 (NHE3) is a brush border (BB) Na+/H+ antiporter that accounts for the majority of physiologic small intestinal and renal Na+ absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins Na+/H+ exchanger regulatory factor (NHERF)1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying lysophosphatidic acid (LPA) stimulation of NHE3 in polarized renal proximal tubule opossum kidney (OK) cells that occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by epidermal growth factor receptor (EGFR), Rho/Rho-associated kinase (ROCK), and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3 μM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKCδ dependent. PKCδ was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA-induced translocation to the membrane of PKCδ was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCδ, which dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility.

The coating for slide plate was prepared using Flake graphite and antioxidant as starting materials. The effects of their additions for the polyethylene glycol (peg) 6000 on the performance of the lubricant were studied, The Oxidation property of the prepared lubricant was compared with the imported lubricant from Japan. The results show that, when the polyethylene glycol 6000 quality percentage content is more than 3%, coating stability, adhesivity, oxidative stability is good. In order to meet the slide coating performance and easy coated, when polyethylene glycol 6000 quality percentage content is 3%, slide paint the best performance.

WO3/TiO2 composite photocatalyst was prepared from tetrabutyl titanate, ammonium tungstate by sol-gel method. The influence of acetic acid and ethanol on the gel point time, and the influence of doping amount of W on the photocatalytic activity was studied. The properties of powders were characterized by Fourier transform infrared spectrometer(FT-IR), and the photocatalytic activity was characterized by probe reaction of methyl orange degradation. The results show that the homogeneous transparent precursor is prepared at the amount of acetic acid =1mL and the amount of ethanol =10mL.When the doping amount of W reached to 0.12%, the best photocatalysis efficiency was obtained. Under the experimental conditions, the infrared absorption band of the W－O was not observed.

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Background & Aims Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na + /H + hydrogen exchanger NHE3 and is increased by Na + -glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood. Methods Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 ( NHERF2−/− mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy. Results The nonmetabolized SGLT1 substrate α-methyl-D-Glu (α-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. α-MD-G normalized NHE3 activity after cholera toxin–induced diarrhea. α-MD-G–stimulated jejunal NHE3 activity was defective in NHERF2−/− mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin–binding domain-dependent increase in co-precipitation of ezrin with NHE3. Conclusions SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin–induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

Six DNA components of Banana bunchy top virus (BBTV) were amplified by polymerase chain reaction (PCR) using total DNA from banana tissues having the typical BBTV symptoms as template. The sequence analysis indicated that the six fragments (1104 nt, 1067 nt, 1059 nt, 1045 nt, 1014 nt and 1081 nt) were all full-length components of BBTV. The viral sequences from different countries were compared. Results revealed that the sequence of component 1 was more conservative than other components, DNA1 of the Hainan isolate shared 95.4% homology with that of the Vietnam isolate, while sequences of both components 2 and 4 showed less homology. DNA2 of the Hainan isolate only shared 83% homology with that of the Guangzhou isolate. According to the results of the DNA1 sequence comparison, the BBTV Hainan isolate should belong to the Asian sub-group.

Activating IK1 channels is considered to be a promising antiarrhythmic strategy. Zacopride has been identified as a selective IK1 channel agonist and can suppress triggered arrhythmias. Whether this drug also exerts a beneficial effect on cardiac remodeling is unknown, and the present study sought to address this question. Cardiac remodeling was induced through coronary ligation-induced myocardial infarction (MI) in male Sprague-Dawley rats. Zacopride (15 µg/kg) was administered (intraperitoneally) daily for 28 days after MI to determine whether it could attenuate MI-induced cardiac remodeling. A 4-week treatment with zacopride attenuated post-MI cardiac remodeling, as shown by the reduced left ventricular end-diastolic dimension and left ventricular end-systolic dimension and the increased ejection fraction and fractional shortening in zacopride-treated animals compared with animals treated with vehicle (all P < 0.05). Furthermore, zacopride significantly decreased myocardial collagen deposition, cardiomyocyte hypertrophy, the plasma level of brain natriuretic peptide, and cardiomyocyte ultrastructural injury. Zacopride also upregulated the expression of the IK1 channel protein and downregulated the expression of phosphorylated p70S6 kinase (p-p70S6K) and mTOR. These beneficial effects of zacopride were partially abolished by the IK1 channel blocker chloroquine. We conclude that the activation of IK1 channel by zacopride attenuates post-MI cardiac remodeling by suppressing mTOR-p70S6 kinase signaling.

In China, porcine blood has a long history as a food with medicinal effects and for treating strokes, an obvious clinical consequence of hypertension. However, the active ingredients are still unknown and need to be clarified. This study described the isolation, characterization and animal experiments of angiotensin converting enzyme (ACE)-inhibitor peptides derived from porcine blood. The highly active low-molecular-weight hydrolysates were obtained from pepsin digestion of discolored porcine blood. After isolation of the hydrolysate, an active fraction containing three peptides was obtained. These peptides were analyzed by MALDI-TOF-MS. They were WVPSV (P1), YTVF (P2), VVYPW (P3), with median inhibitory concentrations of 0.368, 0.226, 0.254 mg/ml, respectively. They were first found in porcine blood. Additionally, a digestion test was performed to verify the antihypertensive effect of the peptides in vitro. After digestion with gastrointestinal proteases, the ACE-inhibitor activity of these peptides was enhanced. When these peptides were administered orally to spontaneously hypertensive rats at a dose of 10 mg/kg, a temporary antihypertensive activity was observed at 3 and 15 h after administration. These findings suggested that the peptides in porcine blood might have potential as antihypertensive agents.

The brush border (BB) Na(+)/H(+) exchanger NHE3 is rapidly activated or inhibited by changes in trafficking, which mimics renal and intestinal physiology. However, there is a paradox in that NHE3 has limited mobility in the BB due to its binding to the multi-PDZ domain containing the NHERF family. To allow increased endocytosis, as occurs with elevated intracellular Ca(2+), we hypothesized that NHE3 had to be, at least transiently, released from the BB cytoskeleton. Because NHERF1 and -2 are localized at the BB, where they bind NHE3 as well as the cytoskeleton, we tested whether either or both might dynamically interact with NHE3 as part of Ca(2+) signaling. We employed FRET to study close association of NHE3 and these NHERFs and fluorescence recovery after photobleaching to monitor NHE3 mobility in the apical domain in polarized opossum kidney cells. Under basal conditions, NHERF2 and NHE3 exhibited robust FRET signaling. Within 1 min of A23187 (0.5 μm) exposure, the NHERF2-NHE3 FRET signal was abolished, and BB NHE3 mobility was transiently increased. The dynamics in FRET signal and NHE3 mobility correlated well with a change in co-precipitation of NHE3 and NHERF2 but not NHERF1. We conclude the following. 1) Under basal conditions, NHE3 closely associates with NHERF2 in opossum kidney cell microvilli. 2) Within 1 min of elevated Ca(2+), the close association of NHE3-NHERF2 is abolished but is re-established in ∼60 min. 3) The change in NHE3-NHERF2 association is accompanied by an increased BB mobile fraction of NHE3, which contributes to inhibition of NHE3 transport activity via increased endocytosis.

Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 degrees C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.

was measured by luciferase reporter activity assays after transiently transfecting Caco-2 cells with DRA promoter construct (-1183/+144). Treatment of Caco-2 cells with 10 μM concentration of ATRA significantly induced DRA mRNA levels within 8 h of incubation, which increased to ~3 fold by 24 h. A concomitant increase (140 ± 5 % of control P ,0.001) in DRA protein expression was also observed. In addition, ATRA treatment significantly increased DRA promoter activity (258.8 ± 3.4 % of control, P,0.001) indicating that ATRA effects are transcriptionally mediated. ATRA is known to mediate its effects via specific retinoic acid receptors (RAR's) α, β and γ. A remarkable increase (~11 fold) in mRNA levels of RAR-β receptor subtype were found in response to ATRA treatment with no change in levels of RAR'-α and γ. Treatment of cells with RAR-β agonist (CH-55, 1μM) mimicked ATRA effects on DRA promoter. RAR-β antagonist (LE-135, 1μM) blocked the stimulatory effects of ATRA on DRA promoter activity. Since, DRA expression is known to be modulated by hepatic nuclear factors (HNF's), the potential mediation of ATRA effects via HNFs was examined. A significant increase in HNF-1β mRNA levels (but not HNF-1α or HNF4) in response to ATRA treatment was observed. The ability of ATRA to induce DRA expression was blocked by silencing HNF-1β using siRNA. In summary, ATRA increased DRA expression via RAR-β through HNF-1β activation. We speculate that ATRA may act as a potential therapeutic tool under inflammatory conditions where DRA expression is altered (Supported by NIDDK and Dept. of Veteran Affairs).

of NHE3 from the membrane and diarrhea. Aim: We investigated the expression, brush border membrane (BBM) localization, and transport function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as the expression of its regulatory PDZ-adapter proteins NHERF1 and PDZK1, TNF-alpha, actin, and a variety of housekeeping genes, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis in the acute phase of diarrhea. Results: After colitis induction, TNF-alpha levels were increased both in the inflamed colon and microscopically normal-appearing small intestine. NHE3 mRNA expression was up regulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid-activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption In Vivo was decreased in the small intestine. PDZK1 mRNA expression was down regulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption. In order to investigate whether the down regulation of PDZK1 and the disturbed NHE3 transport activity is causally related, we studied PDZK1 protein expression and NHE3 transport activity in colonic surface enterocytes in the intestine of PDZK1 +/mice, and found PDZK1 content reduced by 60% and NHE3 transport activity significantly decreased. Conclusion: We suggest that during immune-mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 down regulation during inflammation may be one factor responsible for inflammation-associated NHE3 dysfunction.