Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

氨基酸转运载体SLC38A1研究进展

氨基酸转运载体是介导氨基酸跨膜转运的膜蛋白,在医学、营养等生命科学领域有重要的研究意义。氨基酸转运载体SLC38A1选择性、生理性表达于人体正常大脑和胎盘组织,研究表明,SLC38A1在恶性肿瘤中呈过表达,可以促进肿瘤细胞的增殖、侵袭和迁移。SLC38A1有望成为新的肿瘤靶点之一,本文就SLC38A1在肿瘤中的研究进展作一综述。     

Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan^® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

Bioimaging of multiple elements by high‐resolution LA‐ICP‐MS reveals altered distribution of mineral elements in the nodes of rice mutants

Inter‐vascular transfer in rice (Oryza sativa) nodes is required for delivering mineral elements to developing tissues, which is mediated by various transporters in the nodes. However, the effect of these transporters on distribution of mineral elements in the nodes at a cellular level is still unknown. Here, we established a protocol for bioimaging of multiple elements at a cellular level in rice node by laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS), and compared the mineral distribution profile between wild‐type (WT) rice and mutants. Both relative comparison of mineral distribution normalized by endogenous ¹³C and quantitative analysis using spiked standards combined with soft ablation gave valid results. Overall, macro‐nutrients such as K and Mg were accumulated more in the phloem region, while micro‐nutrients such as Fe and Zn were highly accumulated at the inter‐vascular tissues of the node. In mutants of nodal Zn transporter OsHMA2, Zn localization pattern in the node tissues did not differ from that of WT; however, Zn accumulation in the inter‐vascular tissues was lower in uppermost node I but higher in the third upper node III compared with the WT. In contrast, Si deposition in the mutants of three nodal Si transporters Lsi2, Lsi3 and Lsi6 showed different patterns, which are consistent with the localization of these transporters. This improved LA‐ICP‐MS analysis combined with functional characterization of transporters will provide further insight into mineral element distribution mechanisms in rice and other plant species.     

Structural Characterization of Biomolecules through Atomistic Simulations Guided by DEER Measurements

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主要组织相容性复合体单倍型鸭抗原处理相关转运体单抗的制备

目的制备鸭抗原处理相关转运体(TAP)特异性单抗,为深入利用实验鸭开展免疫学研究提供实验材料。方法利用大肠埃希菌诱导表达主要组织相容性复合体(MHC)单倍型HBW-SPF鸭TAP蛋白肽结合区片段,表达产物经镍柱纯化后免疫BALB/c小鼠,采用ELISA技术筛选特异性单抗分泌杂交瘤细胞株。将阳性细胞株制备小鼠腹水,作为一抗,与多次截短表达TAP蛋白肽结合区进行蛋白免疫印迹试验,鉴定单抗针对的抗原表位。通过间接免疫荧光试验比较该单抗对实验鸭和鸡外周血淋巴细胞的反应性,利用免疫组织化学技术检测对SPF鸡、SPF鸭、鹌鹑、鹅和SPF猪的特异性。结果获得一株鸭TAP单抗1A6,抗原表位位于²⁹⁷NARHQMLQQAVLDATAGTGMVVQEAI³²²,对鸡和鸭外周血淋巴细胞具有免疫荧光反应性;在鸡和鸭的肠黏膜固有层检测到大量特异性信号,在猪、鹌鹑和鹅没有检测到信号。结论获得了一株具有鸡和鸭反应性的抗原转运相关体特异性的单抗,可运用于禽类实验动物在禽病学和禽免疫学方面的研究。     

马铃薯硝态氮转运蛋白基因的克隆及表达分析

该研究以马铃薯双单倍体‘DM’为材料,克隆到高亲和性硝态氮转运蛋白基因StNRT2.1的全长cDNA(JGI登录号PGSC0003DMT400002924),并对其进行表达模式和生物信息学分析,为深入探索StNRT2.1基因的生物学功能以及提高马铃薯对氮素的利用效率奠定理论基础。结果表明:(1)通过同源克隆与PCR扩增获得StNRT2.1基因cDNA全长片段,并构建pCEGFP-StNRT2.1表达载体;测序结果显示其实际所编码的蛋白质序列与数据库中目的基因蛋白质序列完全一致,表明成功克隆到StNRT2.1基因且未出现错义突变。(2)StNRT2.1基因位于马铃薯第11号染色体,cDNA序列全长1 593 bp,编码530个氨基酸,预测蛋白相对分子质量约为57.60 kD,理论等电点为9.36。(3)生物信息学分析显示,StNRT2.1由20种氨基酸组成,其中甘氨酸(Gly)所占比例最多,达到10.8%,并且主要由228个α-螺旋、27个β-折叠、87个延伸链和188个无规则卷曲构成;StNRT2.1存在功能保守结构MFS_1(PF07690)和12个跨膜螺旋结构域,且N端和C端均位于细胞膜内; StNRT2.1位于质膜上且不具有信号肽,可能为非分泌型膜蛋白。(4)以氮充足(7.5 mmol/L)水平作为对照,马铃薯幼苗经无氮(0 mmol/L)和低氮(0.75 mmol/L)处理3周后呈现出叶片发黄及植株矮化等明显表型差异。(5)qRT-PCR结果显示,在无氮条件下,马铃薯根组织中StNRT2.1基因表达量升高3.98倍,说明StNRT2.1可能为诱导型高亲和转运蛋白。     

矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究

硝酸盐转运蛋白（nitrate transporter,NRT）是植物识别、吸收和转运硝酸盐的关键蛋白,对促进作物根系发育、提高产量具有重要作用。通过筛选水生植物,利用NRT蛋白的保守区设计简并引物,并通过PCR和RACE技术,首次从矮珍珠（Glossostigma elatinoides）中克隆得到GeNRT2.1基因。进化分析结果表明,GeNRT2.1与烟草NRT2.1在进化关系上距离最近。qRT-PCR结果表明,GeNRT2.1在矮珍珠根中表达量最高,其次是叶和茎,此外,低浓度硝酸盐（0.5 mmol·L^-1）处理后,GeNRT2.1在根、叶、茎中的表达量分别是高浓度硝酸（2 mmol·L^-1）处理后的1.89、1.93和2.07倍。功能互补实验发现,GeNRT2.1能使缺陷型酵母Δynr恢复生长,具有硝酸盐转运蛋白的功能。通过丰富NRT基因资源,以期为培育氮肥高效利用转基因作物,发展绿色农业,保证我国的粮食安全和环境安全提供理论依据。     

P-glycoprotein models of the apo and ATP-bound states based on homology with Sav1866 and MalK

We exploit the biochemical and sequence similarity between Staphylococcus aureus Sav1866 and P-glycoprotein to develop a homology model of P-glycoprotein representing an ATP-bound state, which captures the major features of the low-resolution EM structure and is consistent with cysteine mutagenesis studies. Using insights from the MalK crystal structures and BtuCD simulations, we model two nucleotide-free conformations. Conformational changes are characterized by pincering rigid-body rotations of the nucleotide-binding domains, inducing transmembrane domain reorganizations which correspond to the two lowest frequency normal modes of the protein. These conformations (see supplementary material) may characterize some of the major steps in the nucleotide catalytic cycle.