Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

重组大肠杆菌全细胞催化L-苏氨酸合成2,5-二甲基吡嗪

2,5-二甲基吡嗪(2,5-dimethylpyrazine,2,5-DMP)在食品香料与医药方面具有重要的经济价值,工业上普遍采用环境不友好且反应条件苛刻的化学合成法来生产.文中结合代谢工程和辅因子工程策略设计高效催化L-苏氨酸合成2,5-DMP的全细胞催化剂,实现微生物转化法合成2,5-DMP.本研究首先分析了不同...     

Nucleotide Binding Domain Interactions During the Mechanochemical Reaction Cycle of ATP-Binding Cassette Transporters

ATP-binding cassette (ABC) transporters serve as importers and exporters for a wide variety of solutes in both prokaryotes and eukaryotes, and are implicated in microbial drug resistance and a number of significant human genetic disorders. Initial crystal structures of the soluble nucleotide binding domains (NBDs) of ABC transporters, while a significant step towards understanding the coupling of ATP binding and hydrolysis to transport, presented researchers with important questions surrounding the role of the signature sequence residues, the composition of the nucleotide binding sites, and the mode of NBD dimerization during the transport reaction cycle. Recent studies have begun to address these concerns. This mini-review summarizes the biochemical and structural characterizations of two archaebacterial NBDs from Methanocaldococcus jannaschii, MJ0796 and MJ1267, and offers current perspectives on the functional mechanism of ABC transporters.     

Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: Heme binding by an induced fit mechanism

Shigella dysentriae and other Gram‐negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB‐dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C‐terminal domain that folds into a 22‐stranded transmembrane β‐barrel, which is filled by the N‐terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA‐Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N‐terminal TonB‐box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB‐box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB‐box. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

一步法RT-PCR克隆水稻线粒体磷转运蛋白基因及其序列特征分析

以水稻广亲和品种Cpslo17幼穗为材料,用一步法RT—PCR（逆转录聚合酶链式反应）克隆了一个长度为1118bp的编码线粒体磷转运蛋白的OsMPT基因。序列分析表明其包含了基因完整的编码序列,编码由368个氨基酸组成的线粒体磷转运蛋白,它与玉米、大豆、Lotus japonicus、Betula pendula、拟南芥的线粒体磷转运蛋白氨基酸序列相似率分别为93．5％,85．6％,83．8％,83．7％,81．1％。氨基酸疏水谱分析显示它有线粒体磷转运蛋白家族高度保守的6个跨膜结构域。水稻线粒体磷转运蛋白N端富含精氨酸（Arginine）、丙氨酸（Alanine）和丝氨酸（Serine）。iPSORT预测其蛋白N端具有定位于线粒体的信号肽序列,进一步分析表明此编码区段有6个外显子和5个内含子。RT—PCR结果表明,OsMPT基因在水稻两个亚种粳稻和籼稻的叶片中均有表达,在Cpslo17营养器官和生殖器官中都有高水平表达。水稻线粒体磷转运蛋白的克隆和表达分析将为研究其结构和生物学功能奠定基础。     

Dopamine Transporter Is Required for In Vivo MPTP Neurotoxicity: Evidence from Mice Lacking the Transporter

Abstract: The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29-fold increase in the level of GFAP mRNA in the striatum of wild-type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP⁺; 10 m M ), via a microdialysis probe produced a massive efflux of DA in wild-type mice (>320-fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.     

The Role of Half-Transporters in Multidrug Resistance

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.     

两种A类氨基酸转运蛋白部分功能差异的研究

应用重组Vaccinia病毒表达系统在猴肾成纤维细胞CV—1中瞬时表达大鼠A类氨基酸转运蛋白GlnT和SAT—2,研究它们转运MeAIB、Ala和Gln的动力学和对多种氨基酸转运亲和性的差异,结合它们的氨基酸序列同源性比较表明：虽然GlnT和SALT—2蛋白的氨基酸序列有较高的同源性,但它们在转运功能上有较大的差异。