Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.     

Mutational analysis and localization of the inositol transporters of Saccharomyces cerevisiae

Saccharomyces cerevisiae possesses two inositol transport proteins, Itr1p and Itr2p, encoded by the ITR1 and ITR2 genes, respectively. Itr1p and Itr2p are high and low affinity transporters, respectively. Eight out of nine cysteine residues in Itr1p, which are common in two transporters, were converted to serine residues by site directed mutagenesis. All mutant genes suppressed the growth defect caused by itr1 disruption, indicating that cysteine residues are not essential for its function. Chimeric genes that express Itr1p and Itr2p fused to the green fluorescent protein (GFP) under the control of the ADH1 promoter were constructed. Both genes were functional. Fluorescence microscopy analysis indicated that both GFP-fused Itr1p and Itr2p are localized to the plasma membrane. A multi-copy plasmid that expresses GFP-fused Itr1p under the control of the original ITR1 promoter was constructed. Under inositol-free culture conditions, GFP-fused Itr1p appeared and was localized to the plasma membrane. When the cells were cultured in the presence of inositol, GFP-fused Itr1p gradually disappeared from the plasma membrane, the fluorescence being redistributed within the cell. Prolonged culture of the cells also caused the relocalization of transporter proteins. These results clearly indicate that the cellular relocalization of transport proteins is responsible for a reduction of inositol transport activity, which is caused by the presence of inositol in the medium or culturing of cells to the stationary phase.     

Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive K(+) transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells.     

Maternal and fetal amino acid concentrations and fetal outcomes during pre-eclampsia

The pathophysiology of pre-eclampsia is contested, but one hypothesis indicates that it is a heterogeneous condition in which only a subset of affected women bear small-for-gestational age (SGA) babies. In intrauterine growth-restricted (IUGR) infants, placental transport of amino acids is diminished and the resulting decrease in cord-blood amino acid concentrations is thought to contribute to their stunted growth. In contrast, the metabolic syndrome (dyslipidaemia, hyperinsulinaemia, hyperglycaemia, hypertension and obesity) which is associated with high amino acid concentrations is more prevalent in women with pre-eclampsia. The focus of this study was to compare maternal and fetal serum amino acid concentrations during normal pregnancy and pre-eclampsia and to evaluate the associations between the amino acid concentrations and fetal growth. The results indicate that maternal and cord-blood amino acid concentrations were significantly higher in women with pre-eclampsia compared with normal pregnant women and the concentrations were inversely associated with measures of infant growth. Maternal and cord-blood amino acid concentrations were also significantly higher in pre-eclamptic mothers with SGA infants compared with pre-eclamptic mothers whose babies were not SGA. These data indicate that, in contrast to IUGR, pre-eclampsia is associated with enhanced placental amino acid transport or reduced fetal amino acid utilization. Furthermore, the data are consistent with the hypothesis that pre-eclampsia is a heterogeneous disease associated with the metabolic syndrome.     

谷氨酸棒杆菌的氨基酸输送系统的存在、功能及其在氨基酸生产上的重要性

主要介绍了谷氨酸棒杆菌(Corynebacteriumglutamicum)中各种氨基酸输送系统及其基因、底物、特性和在氨基酸生产上的应用.     

植物中花色苷转运蛋白研究进展

花色苷是植物重要的次生代谢产物,因其具有高抗氧化活性受到广泛关注。目前,关于花色苷合成代谢途径及相关转录调控机制已有了较为深入的研究,其相关结构基因及关键转录调控因子已在多种植物中被陆续鉴定与验证,但对于花色苷合成后的运输机制的认识仍相对缺乏。花色苷合成主要发生在内质网中,随后运送至液泡内进行储存。花色苷的运输过程主要包括囊泡运输及链接蛋白运输两种模式,而转运蛋白在两种运输模式中均发挥重要作用。鉴定花色苷转运相关蛋白有助于深入了解花色苷的运输机制,完善对花色苷代谢途径的认知。本文主要针对谷胱甘肽S-转移酶(glutathione S-transferase,GST)、ATP-结合框(ATP-binding cassette,ABC)转运蛋白、多药和毒性化合物排出转运蛋白(multidrug and toxic extrusion compound transporters,MATE)和胆红素异位酶(bilitranslocase,BTL) 4类花色苷转运相关蛋白的研究进展进行了概述。     

烟草祖先种及重要野生种PDR1基因的生物信息学分析

植物的多向耐药性（Pleiotropic drug resistance, PDR）转运蛋白隶属于ATP结合盒（ATP-binding cassette, ABC）转运蛋白家族的G亚家族。该类蛋白参与植物多种初生、次生代谢物的跨膜运输,在植物的生长发育、响应逆境胁迫、解毒过程中起着重要作用。近年的研究还表明,该蛋白与多种植物对真菌病原的抗性有关。本文以烟草祖先种和重要野生种为研究对象,以拟南芥PDR12蛋白、烟草的PDR1基因为探针,通过搜索同源序列,从数据库中提取烟草及祖先种、重要野生种中的PDR基因、蛋白序列,并通过生物信息学分析手段对其编码蛋白的理化特性、氨基酸序列特征、蛋白结构、功能、启动子序列及表达调控特征进行了分析,并以本氏烟（Nicotiana benthamiana）NbPDR1为代表,预测了该基因启动子的顺式作用元件,以普通烟草（Nicotiana tabacum）NtPDR1基因为例,预测了其基因编辑位点。结果表明,这些PDR基因相似性高,均编码跨膜蛋白,具有典型的跨膜结构域和核酸结合结构域。该基因启动子区有多个与病原真菌诱导、干旱等逆境胁迫响应相关的元件,暗示该基因可能是抗真菌病害、响应逆境胁迫的广谱抗性基因。最后,本文预测的基因编辑位点,为烟草PDR基因的功能研究、基因编辑及抗病育种提供理论基础。      

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.     

Active intestinal absorption of nucleosides by Na+-dependent transport across the brush border membrane in cows

Transport of 3H-labeled nucleosides across the bovine intestinal brush border membrane (BBM) was characterized with BBM vesicles (BBMV) isolated from mid-jejunum of cows because large amounts of nucleic acids are digested in the small intestine of ruminants. Two Na+-dependent electrogenic nucleoside transporters with overlapping substrate specificity were shown to be present in the jejunal BBM, one for pyrimidine nucleosides and one for purine nucleosides. As indicated by inhibitory studies, thymidine seemed to be a specific substrate for the pyrimidine nucleoside transporter, while this applied to guanosine and deoxyguanosine for the purine nucleoside transporter. Uridine and adenosine appear to have an affinity to both transporters. This also applies to deoxyadenosine and deoxyuridine. Nucleobases (uracil, hypoxanthine) did not affect transport of nucleosides. The kinetic constants (Km and Vmax) for Na-dependent thymidine and guanosine transport were 29 and 24 micromol/L and 78 and 51 pmol (mg protein)(-1) s(-1), respectively. These values are much higher than those reported for monogastric species.     

色氨酸对断奶仔猪氨基酸转运载体的影响

色氨酸(tryptophan,Trp)是一种特殊的氨基酸,它参与调节蛋白质的合成,同时在调节食欲与维持等方面都有很重要的作用。本文通过测定断奶仔猪的生长性能、血清中生化指标和游离氨基酸含量、肝脏色氨酸2,3-加双氧酶(tryptophan 2,3-dioxygenase,TDO)和肠道吲哚胺2,3-加双氧酶(indoleamine 2,3-dioxygenase,IDO)含量及Trp相关氨基酸转运载体基因表达,来探讨Trp对断奶仔猪氨基酸转运载体的影响。结果发现,添加0.2%Trp可显著增加日增重与日采食量,显著增加血液中色氨酸与亮氨酸的含量,显著降低缬氨酸与异亮氨酸的含量。0.1%Trp可显著增强肝脏TDO含量,0.2%Trp显著增加空肠IDO含量。添加0.2%Trp显著增加空肠基因ATB0,+、4F2hc、B0AT1和ASCT2表达(P0.05)。综上,Trp能够促进相关氨基酸转运载体的表达,最终促进氨基酸的转运吸收。说明Trp可以通过上调氨基酸转运载体表达来调控机体健康。     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast. Copyright © 2015 John Wiley & Sons, Ltd.     

Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae

To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters. Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity. Disruption of ADY2 in S. cerevisiae abolished the active transport of acetate. Microarray analyses of ady2Delta strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport. Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate.     

Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (K(m)) of 9.8 ± 3.0mM for 2-deoxy-d-glucose, similar to 11.7 ± 3.7 mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.     

烤烟烟碱转运蛋白基因与烟碱积累的相关性研究

解析烟碱转运相关基因对烟碱积累的影响,可为定向改良烟碱性状提供理论指导。本研究对7份烟碱含量不同的烤烟进行水培,检测打顶前后烟碱含量、烟碱合成基因（NtPMT、NtQPT）、烟碱转运基因（NtNUP1、NtJAT1/2、NtMATE1/2）以及囊泡运输调节基因（NtGEF）的表达量变化。经相关分析、主成分分析和相关网络分析表明：（1）烟碱合成基因NtPMT、NtQPT与烟碱转运基因NtNUP1、NtMATE1/2相互促进表达;（2）根部和叶部的主效烟碱转运基因中,NtJAT1是腰叶的主要烟碱转运基因,NtNUP1、NtJAT1、NtMATE1/2在根部对烟碱转运都有贡献,且转运效率依次递减;（3）囊泡运输调节因子NtGEF也能间接影响叶部烟碱积累。因此,在对烟碱性状进行定向改良时,可考虑烟碱合成、转运以及囊泡运输调节因子对烟碱积累的贡献大小。     

基于宏基因组分析酸马奶的微生物多样性及功能基因

酸马奶风味独特、保健功能突出,这与其复杂的微生物构成密切相关.采用宏基因组技术分析酸马奶的微生物多样性,挖掘其功能基因.结果表明:从酸马奶中鉴定出的微生物分属于30个门,331个科,913个属,2692个种.优势菌种为马乳酒样乳杆菌、瑞士乳杆菌、弗氏柠檬酸杆菌、解鸟氨酸拉乌尔菌、柠檬酸杆菌和乳酸乳球菌.在COG、KEG...     

Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.