ABC转运蛋白基因slnTI和slnTII与盐霉素生物合成的相关性

【目的】slnTI和slnTII是盐霉素生物合成基因簇中可能的两个转运蛋白基因,根据生物信息学的分析推测它们属于ABC转运蛋白家族。其中,slnTI编码ABC转运蛋白的ATP结合亚基,slnTII编码ABC转运蛋白的跨膜亚基,推测它们可能与盐霉素的外排有关。通过slnTI和slnTII的基因中断与超量表达研究它们对盐霉素生物合成产量和抗性的影响。【方法】利用REDIRECT?技术,在盐霉素产生菌白色链霉菌XM211中分别构建了slnTI和slnTII的基因置换突变株LJ01和LJ02,并通过基因回补对突变株进行了验证。利用整合型表达载体pPM927在白色链霉菌XM211中对slnTI和slnTII进行串联超量表达。将slnTI和slnTII导入变铅青链霉菌1326中进行异源表达,通过液体培养实验检测衍生菌株对盐霉素的抗性。【结果】相比出发菌株XM211,突变株LJ01中盐霉素的产量下降了27.2%,LJ02下降了45.4%,LJ01和LJ02中结构基因slnA3和调控基因slnR的转录水平都有明显降低。超量表达菌株LJ03中盐霉素的产量提高了14.6%,转录结果显示LJ03中不仅slnTI和slnTII自身转录水平有大幅提高,而且slnA3和slnR转录水平也显著升高。抗性检测结果表明,异源表达菌株变铅青链霉菌LJ04对盐霉素的抗性水平略有提高。【结论】slnTI和slnTII是与盐霉素生物合成和外排有关的ABC转运蛋白基因,但并不是白色链霉菌XM211对盐霉素的主要抗性基因。     

植物蔗糖转运蛋白表达的调控因素与分子机制

植物光合作用的产物主要以蔗糖的形式在植物体内进行从源到库的运输.蔗糖转运蛋白是此过程的重要参与者,其表达和调控与植物中光合作用产物的分配紧密关联,从而调控着植物的生长发育、结果结实、抗逆抗病等性状.蔗糖转运蛋白的表达受到植物发育时期、外界环境条件及激素的影响.蔗糖转运蛋白的调控机制有转录因子的调节、基因内部序列调控、蛋...     

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.     

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.     

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...     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

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.