LOCALIZATION AND FUNCTIONAL CHARACTERIZATION OF OATP4C1 TRANSPORTER IN \u3ci\u3eIN VITRO\u3c/i\u3e CELL SYSTEMS AND HUMAN/RAT TISSUES

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. In vitro studies in polarized cell lines showed that upon transfection rat Oatp4c1 localizes at the apical membrane. The objectives of this project were to further validate the subcellular localization of Oatp4c1/OATP4C1 in rat and human tissues as well as their localization and function in polarized cells. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunoblotting, we investigated the expression pattern of Oatp4c1 in epithelial cell lines and in the rat kidney and mammary gland (MG). Collectively, these data demonstrated that rat Oatp4c1 localized at the apical cell surface of polarized epithelium and primarily in the proximal straight tubules, the S3 segment of proximal tubule, in the juxtamedullary cortex. Drug uptake studies in Oatp4c1-expressing cells demonstrated that Oatp4c1- mediated estrone-3-sulfate (E3S) uptake was ATP-independent and pH-dependent. The increased E3S transport activity at acidic extracellular pH was ascribed to the increased maximum transport rate (Vmax). In addition, E3S transport inhibition by various substrates suggests that Oatp4c1 possesses multiple substrate binding sites. The apical localization of Oatp4c1 in the rat kidney and MG is a novel finding and implies that this transporter protein plays a role in the reabsorption, not vectorial secretion, of its substrates. In addition, the upregulation of Oatp4c1 expression during lactation indicates that it is involved in reuptake of xenobiotic from the milk, resulting in their reduced exposure to the suckling infants, or that it functions as a scavenger system. Further, studies to identify physiological substrates are needed to better understand the significance of Oatp4c1 function in renal and mammary epithelium

Hospital treatment, mortality and healthcare costs in relation to socioeconomic status among people with bipolar affective disorder

BACKGROUND: Evidence regarding the relationships between the socioeconomic status and long-term outcomes of individuals with bipolar affective disorder (BPD) is lacking. AIMS: We aimed to estimate the effects of baseline socioeconomic status on longitudinal outcomes. METHOD: A national cohort of adult participants with newly diagnosed BPD was identified in 2008. The effects of personal and household socioeconomic status were explored on outcomes of hospital treatment, mortality and healthcare costs, over a 3-year follow-up period (2008–2011). RESULTS: A total of 7987 participants were recruited. The relative risks of hospital treatment and mortality were found elevated for the ones from low-income households who also had higher healthcare costs. Low premium levels did not correlate with future healthcare costs. CONCLUSIONS: Socioeconomic deprivation is associated with poorer outcome and higher healthcare costs in BPD patients. Special care should be given to those with lower socioeconomic status to improve outcomes with potential benefits of cost savings in the following years. DECLARATION OF INTEREST: None. COPYRIGHT AND USAGE: © 2016 The Royal College of Psychiatrists. This is an open access article distributed under the terms of the Creative Commons Non-Commercial, No Derivatives (CC BY-NC-ND) licence

Hospital treatment, mortality and healthcare costs in relation to socioeconomic status among people with bipolar affective disorder

BACKGROUND: Evidence regarding the relationships between the socioeconomic status and long-term outcomes of individuals with bipolar affective disorder (BPD) is lacking. AIMS: We aimed to estimate the effects of baseline socioeconomic status on longitudinal outcomes. METHOD: A national cohort of adult participants with newly diagnosed BPD was identified in 2008. The effects of personal and household socioeconomic status were explored on outcomes of hospital treatment, mortality and healthcare costs, over a 3-year follow-up period (2008–2011). RESULTS: A total of 7987 participants were recruited. The relative risks of hospital treatment and mortality were found elevated for the ones from low-income households who also had higher healthcare costs. Low premium levels did not correlate with future healthcare costs. CONCLUSIONS: Socioeconomic deprivation is associated with poorer outcome and higher healthcare costs in BPD patients. Special care should be given to those with lower socioeconomic status to improve outcomes with potential benefits of cost savings in the following years. DECLARATION OF INTEREST: None. COPYRIGHT AND USAGE: © 2016 The Royal College of Psychiatrists. This is an open access article distributed under the terms of the Creative Commons Non-Commercial, No Derivatives (CC BY-NC-ND) licence

Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature

Prediction of molecular transport pathways in the human glucose transporter 1 (GLUT1)

醣類是身體重要的能源之一，但由於其水溶性的特性，需要透過細胞膜表面的特別蛋白通道，才能穿過脂質所構成的細胞膜來被細胞吸收利用。這個運輸通道對於葡萄糖具有獨特的專一性，只允許葡萄糖分子自由穿越細胞膜。葡萄糖運轉蛋白家族(GLUT family)在身體各組織細胞中細胞的表現數量不同，且運輸葡萄糖時的速度也不同，以此達到體內葡萄糖濃度的恆定。這些蛋白被稱為GLUT家族（GLUT family），依其被發現的順序被分別命名為GLUT1、GLUT2、GLUT3、GLUT4……依此類推。其中GLUT1主要分佈在紅血球，是目前被廣泛研究的細胞膜蛋白(membrane transporter)之一。但經由GLUT1運輸葡萄糖的分子機制目前仍有許多未被瞭解。 由於膜蛋白的結晶不易，至今仍未有實驗方法可以得到其高解析度的結構。因此以大腸桿菌的glycerol phosphate transporter為模本及glucose 6-phosphate translocase為中間模本，利用同源模擬的方法建立初始結構。再將此初始結構放到細胞膜的原子模型中，加入足量的水分子將其完整包覆，並調整成0.15M氯化鈉的生理環境，利用分子動力學模擬方法修飾此初始結構。根據化學交聯(chemical crosslinking)實驗結果，我們將G145及V328間的距離漸漸修飾調整到16 Å後，再移除此拉力並繼續模擬，使蛋白質可以回到其最喜歡且穩定的構形。利用umbrella sampling方法以及weighted histogram analysis method (WHAM)以計算得出平均力場(potential of mean force)，由結果可看出修飾過後的結構的自由能的確較低，G145 及 V328間的最佳距離是18 Å，且S148-V328, G145-L325 及 S148-L325在拉力移除後的模擬過程中，其距離也一直在6-16 Å間，與化學交聯實驗結果吻合。另外由嵌合模擬實驗結果可發現利用修飾完的結構能更準確的預測出葡萄糖結合位置。 利用一個新的嵌合(docking)模擬程序，可以找出一系列相鄰的配體(ligand)結合位置及其較佳的結合構形，如此可預測出葡萄糖運輸途徑(transport pathway)。另外，藉由得到沿著此運輸途徑受質與蛋白質間的結合自由能(binding free energy)可用來探討受質的選擇性。將修飾完的結構結合葡萄糖進行分子動力學模擬計算，以探討在葡萄糖運輸過程中GLUT1的構形變化。對於葡萄糖運輸蛋白的結構與其運輸過程有更清楚的瞭解可以幫助葡萄糖不平衡如乙型糖尿病的藥物開發研究。Passive transport of glucose across the plasma membrane is mediated by members of the glucose transporter (GLUT/SLC2A) family which belongs to the major facilitator superfamily (MFS). GLUT1, known as the red blood cell glucose transporter, is one of the most extensively studied membrane transporters. However, the molecular mechanism of GLUT1-mediated glucose transport is still little clarified. The homology model of GLUT1(PDB code: 1SUK) was constructed using the crystal structure of E. coli glycerol phosphate transporter as the template and glucose 6-phosphate translocase as the intermediate. We have conducted molecular dynamics (MD) simulations of the GLUT1 in the full-hydrated POPC bilayer to refine the homology model based on chemical crosslinking data determined by di-cysteine mutagenesis. The distance between G145 and V328 was gradually restrained to 16 Å by umbrella sampling technique and then the restraint was removed, in this way, the GLUT1 was relaxed to its favorable conformation. The weighted histogram analysis method (WHAM) was employed for calculating the potential of mean force (PMF) profile, which indicated that the refined structure did have lower free energy. The minima of the PMFs located at the same point indicate that the optimal distance between G145 and V328 is around 18 Å. The distances of S148-V328, G145-L325 and S148-L325 after simulation are within 6-16 Å, which is consisted with the chemical crosslinking data. It was found that the predicted glucose binding sites generated from docking simulations on the refined structure were in better agreement with the existent experiments compared to those on the original homology structure. The glucose transport pathways were predicted by a novel docking scheme, SLITHER, which was modified from AutoDock 3.05. It can locate a series of juxtaposed ligand binding sites within such membrane channels, along with the most favorable conformation at each binding site. The binding free energies of substrates along the pathways can be used to investigate the mechanisms of substrate selectivity. The MD simulations of the GLUT1 with glucose in a full-hydrated POPC bilayer were conducted. The simulation results can be used to investigate the conformational changes upon glucose transport. Detailed knowledge of the structure of the glucose transporters will lead to advances in the understanding and therapeutics of glucose homeostasis disorders, including type 2 diabetes mellitus.Table of Content i Abstract iii 中文摘要 v Chapter 1 Introduction 1 Chapter 2 Structural Analysis of GLUT1 5 2.1 Alternating Conformational Model 5 2.2 Structure/Function Studies 8 2.3 GLUT1 Deficiency Syndrome 10 2.4 Homology Model 13 2.5 Summary of the Previous Application Using the GLUT1 Homology Model 15 2.6 Analysis of the Proximity of TM4 and TM8 by Chemical Crosslinking 18 Chapter 3 Molecular Dynamics Simulation of the GLUT1 21 3.1 Construction of the GLUT1 in a Full-hydrated POPC Lipid Bilayer 21 3.2 Energy Minimization and Equilibration 24 3.3 Analysis of Molecular Dynamics Simulation 25 Chapter 4 Structure Refinement Based on Chemical Crosslinking data 31 4.1 Structure Refinement by Iterative Umbrella Sampling 31 4.2 Weighted Histogram Analysis Method (WHAM) for Potential of Mean Force (PMF) Calculation 36 Chapter 5 Prediction of the Glucose Transport Pathways 47 5.1 HOLE 47 5.2 SLITHER 50 Chapter 6 Molecular Dynamics Simulations of GLUT1 with Glucose Binding 71 6.1 Docking of Glucose to GLUT1 71 6.2 Analysis of Molecular Dynamics Simulation 72 Chapter 7 Conclusion 103 Reference 10

Alpinia oxyphylla Fruit Extract Ameliorates Experimental Autoimmune Encephalomyelitis through the Regulation of Th1/Th17 Cells

Alpinia oxyphylla is a traditional Chinese medicine widely used for treating diarrhea, ulceration, and enuresis. Moreover, A. oxyphylla is effective for cognitive function improvement and nerve regeneration. Multiple sclerosis (MS) is a chronic neuronal inflammatory autoimmune disease that commonly affects young adults in high-latitude regions. The aim of this study was to evaluate the beneficial effects of A. oxyphylla in an experimental autoimmune encephalomyelitis (EAE) mouse model, which is an extensively used model for human MS. The ethanolic extract of A. oxyphylla fruit (AO-1) was orally administered to EAE mice. Our results showed AO-1 significantly reduced EAE symptoms. Histopathological analysis showed AO-1 reduced demyelination, inflammation, gliosis, and axonal swelling in the spinal cord. Furthermore, immunohistochemistry and quantitative polymerase chain reaction (qPCR) studies revealed that the infiltration of CD4+, CD8+ T cells, and CD11b+ monocytes into the spinal cord decreased in the AO-1-treated group. Mechanistically, the Th1 transcription factor T-bet, Th17 transcription factor retinoic acid receptor–related orphan receptor γ (RORγt), and inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 were reduced in the spinal cords of mice treated with AO-1. The expression levels of T-bet and RORγt were also lowered in the spleens of those mice. Further in vitro study showed AO-1 inhibited production of IFN-γ, IL-2, and tumor necrosis factor-α from MOG35-55-peptide-stimulated splenocytes. One component isolated from AO-1, yakuchinone A, inhibited IL-17 production in vitro and reduced EAE symptoms in the mice. Collectively, our results indicate that AO-1 ameliorated the severity of EAE in mice and may involve the regulation of Th1/Th17 response. A. oxyphylla warrants further investigation, particularly regarding its clinical benefits for MS

Expression of Oatp4c1 in isolated kidney brush border membrane (BBM) and basolateral membrane (BLM) fractions.

<p>(A) Western blot analysis for Oatp4c1 expression in BBM and BLM. Biochemical separation of membrane proteins (20 µg per lane) from rat kidney cortex was achieved by Percoll gradient. Na⁺/K⁺-ATPase alpha 1 was used as a basolateral marker. Abcg2 and Mrp4 served as apical markers. Oatp4c1 was detected by PA1343. (B) LC-MS/MS based proteomic analysis of ∼60–150 kD proteins isolated from BBM and BLM fractions. Spectral counts detected in protein digests obtained from ∼60–150 kD sections excised from the SDS-PAGE gel. Proteins with 3 or more spectral counts in one fraction are presented as “Apical” or “Basolateral” based on previously published data. Spectral counts indicate the number of unique peptides detected in each fraction. (AP, apical).</p

Transport kinetic parameters of Oatp4c1-mediated E3S transport at pH 4.5, 5.5 and 7.4 (mean ± S.E.).

<p>The parameters were estimated by fitting a sigmoid-E_max model to the data. Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test for all pairwise comparisons. *, **, *** p<0.05 indicate significant differences between pH 4.5 and 5.5, pH 4.5 and 7.4, and pH 5.5 and 7.4, respectively.</p