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Cotransfecting norepinephrine transporter and vesicular monoamine transporter 2 genes for increased retention

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《医学前沿（英文）》 2017年第11卷第1期页码 120-128 doi: 10.1007/s11684-017-0501-3

摘要：

Norepinephrine transporter (NET) transfection leads to significant uptake of iodine-131-labeled metaiodobenzylguanidine (¹³¹I-MIBG) in non-neuroendocrine tumors. However, the use of ¹³¹I-MIBG is limited by its short retention time in target cells. To prolong the retention of ¹³¹I-MIBG in target cells, we infected hepatocarcinoma (HepG2) cells with Lentivirus-encoding human NET and vesicular monoamine transporter 2 (VMAT2) genes to obtain NET-expressing, NET-VMAT2-coexpressing, and negative-control cell lines. We evaluated the uptake and efflux of ¹³¹I-MIBG both in vitro and in vivo in mice bearing transfected tumors. NET-expressing and NET-VMAT2-coexpressing cells respectively showed 2.24 and 2.22 times higher ¹³¹I-MIBG uptake than controls. Two hours after removal of ¹³¹I-MIBG-containing medium, 25.4% efflux was observed in NET-VMAT2-coexpressing cells and 38.6% in NET-expressing cells. In vivo experiments were performed in nude mice bearing transfected tumors; results revealed that NET-VMAT2-coexpressing tumors had longer ¹³¹I-MIBG retention time than NET-expressing tumors. Meanwhile, NET-VMAT2-coexpressing and NET-expressing tumors displayed 0.54% and 0.19%, respectively, of the injected dose per gram of tissue 24 h after ¹³¹I-MIBG administration. Cotransfection of HepG2 cells with NET and VMAT2resulted in increased ¹³¹I-MIBG uptake and retention. However, the degree of increase was insufficient to be therapeutically effective in target cells.

关键词： norepinephrine transporter vesicular monoamine transporter 2 -MIBG gene therapy lentivirus vector

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Loss of monocarboxylate transporter 1 aggravates white matter injury after experimental subarachnoid

《医学前沿（英文）》 2021年第15卷第6期页码 887-902 doi: 10.1007/s11684-021-0879-9

摘要： Monocarboxylic acid transporter 1 (MCT1) maintains axonal function by transferring lactic acid from oligodendrocytes to axons. Subarachnoid hemorrhage (SAH) induces white matter injury, but the involvement of MCT1 is unclear. In this study, the SAH model of adult male Sprague-Dawley rats was used to explore the role of MCT1 in white matter injury after SAH. At 48 h after SAH, oligodendrocyte MCT1 was significantly reduced, and the exogenous overexpression of MCT1 significantly improved white matter integrity and long-term cognitive function. Motor training after SAH significantly increased the number of ITPR2⁺SOX10⁺ oligodendrocytes and upregulated the level of MCT1, which was positively correlated with the behavioral ability of rats. In addition, miR-29b and miR-124 levels were significantly increased in SAH rats compared with non-SAH rats. Further intervention experiments showed that miR-29b and miR-124 could negatively regulate the level of MCT1. This study confirmed that the loss of MCT1 may be one of the mechanisms of white matter damage after SAH and may be caused by the negative regulation of miR-29b and miR-124. MCT1 may be involved in the neurological improvement of rehabilitation training after SAH.

关键词： microRNAs monocarboxylate transporter 1 motor training subarachnoid hemorrhage white matter injury

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a novel K+ transporter gene in cotton

Yiru WANG, Juan XU, Mingcai ZHANG, Xiaoli TIAN, Zhaohu LI

《农业科学与工程前沿（英文）》 2018年第5卷第2期页码 226-235 doi: 10.15302/J-FASE-2017170

摘要： Potassium is an essential nutrient for plant growth and productivity of crops. K transporters are important for K uptake and transport in plants. However, information on the function of K transporters and K channels in cotton is limited. The KT/KUP/HAK protein family is essential for a variety of physiological processes in plants, including nutrient acquisition and regulation of development. This study, identified a K transporter gene, expressed in the roots of cotton ( ) cv. Liaomian17. The deduced transcript of is highly homologous to Cluster II of KUP/HAK/KT K transporters and is predicted to contain 11 transmembrane domains. has been localized to the plasma membrane, and its transcripts were detected in roots, stems, leaves and shoot apices of cotton seedlings. Consistently, b-glucuronidase (GUS) expression driven by the GhKT2 promoter could be detected in roots, mesophyll cells, and leaf veins in transgenic . In addition, the expression of was induced by low K stress in cotton roots and ::GUS-transgenic seedlings. The -overexpression lines plants were larger and showed greater K accumulation than the wild type (WT) regardless of K concentration supplied. The net K influx rate, measured by the noninvasive micro-test technique, in root meristem zone of -transgenic lines was significantly greater than that of WT. Taken together, this evidence indicates that GhKT2 may participate in K acquisition from low or high external K , as well as K transport and distribution in plants.

关键词： cotton GhKT2 potassium transporter uptake

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Identification of transporter proteins for PQQ-secretion pathways by transcriptomics and proteomics analysis

Hui Wan,Yu Xia,Jianghua Li,Zhen Kang,Jingwen Zhou

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 72-88 doi: 10.1007/s11705-016-1580-4

摘要： Pyrroloquinoline quinone (PQQ) plays a significant role as a redox cofactor in combination with dehydrogenases in bacteria. These dehydrogenases play key roles in the oxidation of important substrates for the biotechnology industry, such as vitamin C production. While biosynthesis of PQQ genes has been widely studied, PQQ-transport mechanisms remain unclear. Herein, we used both two-dimensional fluorescence-difference gel electrophoresis tandem mass spectrometry and RNA sequencing to investigate the effects of overexpression in an industrial strain of WSH-003. We have identified 73 differentially expressed proteins and 99 differentially expressed genes, a majority of which are related to oxidation-reduction and transport processes by gene ontology analysis. We also described several putative candidate effectors that responded to increased PQQ levels resulting from overexpression. Furthermore, quantitative PCR was used to verify five putative PQQ-transport genes among different PQQ producing strains, and the results showed that , and were upregulated in all conditions. Then the three genes were over-expressed in WSH-003 and PQQ production were detected. The results showed that extracellular PQQ of B932_1930 (a transporter) and B932_2186 (an ABC transporter permease) overexpression strains were enhanced by 1.77-fold and 1.67-fold, respectively. The results suggest that the proteins encoded by PqqB, B932_1930 and B932_2186 might enhance the PQQ secretion process.

关键词： 2D-DIGE pqqB pyrroloquinoline quinone RNA-Seq Vitamin C

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Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

Xixun DU*, Huamin XU*, Hong JIANG, Jun WANG, Lei WANG, Junxia XIE

《医学前沿（英文）》 2009年第3卷第1期页码 67-71 doi: 10.1007/s11684-009-0010-0

摘要： Divalent metal transporter 1 (DMT1) is a ferrous iron import protein. The improper expression of DMT1 is involved in neurodegenerative diseases. In the present study, we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element (DMT1-IRE) and investigated its expression and function in the C6 glioma cell line. The DMT1-IRE gene, obtained by RT-PCR, was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene. Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into ( ) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with I. I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells (HEK293 cells) , in which recombinant adenoviruses were generated within 7 to 10 days. The results demonstrated that we obtained the DMT1-IRE gene. pAdEasy1-DMT1-IRE yielded a large fragment, plus a smaller fragment of 4.5 kb after digestion with I. PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE, indicating the successful construction of recombinant adenovirus plasmid containing DMT1-IRE. GFP fluorescence further confirmed the generation of adenovirus. AdDMT1-IRE could efficiently infect C6 glioma cells. And cell viability decreased in Ad-DMT1-IRE infected cells after iron overload compared to the control. These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.

关键词： divalent metal transporter 1 recombinant adenovirus homologous recombination iron