Polymorphisms in the canine glutamate transporter-1 gene: identification and variation among five dog breeds

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.     

Content of ileal EAAC1 and hepatic GLT-1 high-affinity glutamate transporters is increased in growing vs. nongrowing lambs,paralleling increased tissue D- and L-glutamate,plasma glutamine,and alanine concentrations

Glutamate is a central metabolite for whole-animal energy and N metabolism. This study tested the hypothesis that ileal epithelium, liver, and kidney content of system X-(AG) glutamate transporters EAAC1 and GLT-1 would be up-regulated to support growth of wethers (30 +/- 1.2 kg) fed a forage-based diet for at least 14 d to gain (2.0 x NEm; n = 9) vs. maintain (1.2 x NEm; n = 9) BW. We have previously demonstrated that two high-affinity glutamate transporters (EAAC1, GLT-1) are expressed by these extensive glutamate metabolizing epithelial tissues. Wethers fed at 2.0 x NEm gained (P < 0.001; 0.26 kg/d) BW, whereas those fed 1.2 x NEm did not. Although plasma concentrations (microM) of glucose and L- or D-glutamate did not differ, plasma glutamine (precursor of glutamate) and alanine concentrations (transamination product of glutamate) were 28% (P < 0.007) and 22% (P < 0.072) greater for growing lambs than nongrowing lambs. In tissues, the concentration of L-glutamate in ileum epithelia and D-glutamate of liver was 49% (P < 0.015) and 181% (P < 0.042) greater, respectively, in growing vs. nongrowing animals, whereas concentrations of glutamate isoforms did not differ in kidney. Paralleling these increased amino acid concentrations, ileal epithelium contained 313% more (P < 0.038) EAAC1 protein and liver contained 240% more (P < 0.001) GLT-1 protein, whereas kidney transporter content did not differ between growing and nongrowing wethers. In contrast to increased EAAC1 and GLT-1 protein content in ileal and liver tissue of growing lambs, messenger RNA levels did not differ. These results indicate that the increased capacity for high-affinity glutamate uptake in growing vs. nongrowing lambs is achieved through increased expression of EAAC1 by ileal epithelium and GLT1 by liver, which parallel increased tissue concentrations of glutamate and plasma concentrations of two major interorgan N carriers, glutamine and alanine.     

Transient ischaemia affects plasma membrane glutamate transporter, not vesicular glutamate transporter, expressions in the gerbil hippocampus

In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.     

Comparative studies on the distribution of glutamate transporters in the retinae of the Mongolian gerbil and the rat

Glutamate is the major excitatory amino acid transmitter in vertebrate retinae. Glutamate transporters therefore play an important role in the precise control of glutamate concentration in the synaptic cleft by regulating extracellular glutamate concentration. In the present study, we performed an analysis of the expressions of three glutamate transporters in gerbil retina using immunohistochemistry. In the gerbil retina, excitatory amino acid carrier 1 and glutamate transporter 1 immunoreactivity was predominant in the ganglion cells but not amacrine or bipolar cells. Glutamate/aspartate transporter (GLAST) immunoreactivity was observed in the radial gliocytes of which the dense network of fine processes was localized in the inner and outer plexiform layers. GLAST immunoreactivity was also detected in astrocytes in the nerve fibre layer. These results demonstrate that three glutamate transporters show specific distributions in the gerbil retina and suggest that the glutamate re-uptake system in the gerbil retina may be different from that of the rat.     

早期断奶对仔猪空肠和回肠兴奋性氨基酸载体1表达的影响

本试验旨在研究仔猪出生后10~20 d,早期断奶仔猪小肠谷氨酸转运载体基因表达情况与哺乳仔猪的差异。试验分别从40头不同母猪的仔猪中各选出体重相近,10日龄的"杜×长×大"三元杂交仔猪1头,共40头仔猪,随机不配对分为2组,每组20头仔猪,对照组(哺乳组)为哺乳仔猪,随母猪喂养;试验组(断奶组)为断奶仔猪,隔离断奶饲养;试验期10 d。饲养结束,每组随机取12只仔猪,宰杀取空肠和回肠,测定谷氨酸转运载体兴奋性氨基酸转运载体1(EAAC1)蛋白质表达情况和游离氨基酸含量。结果显示,断奶显著降低了仔猪空肠和回肠EAAC1(57和73 ku)及其相关蛋白谷氨酸转运联合蛋白(GTRAP3-18)(50 ku)的蛋白质和mRNA表达量(P0.05)。断奶提高了仔猪空肠游离谷氨酸和总氨基酸含量,却降低了仔猪回肠游离谷氨酸和总氨基酸含量,差异显著(P0.05)。结果提示,早期断奶降低EAAC1和GTRAP3-18的蛋白质含量,这可能与早期断奶仔猪遭受营养谷氨酸缺乏导致的肠道氨基酸吸收转运障碍有关。     

槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制。猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2%DMSO处理。采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)法测定氨基酸和小肽转运载体以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的mRNA相对表达量;采用Western blot法测定mTOR信号通路相关基因的蛋白表达。结果表明:与对照组相比,1)0.4和0.8 mg/L槲皮素均极显著增加猪肠上皮细胞中蛋白质的含量(P<0.01)。2)1.6 mg/L槲皮素极显著提高猪肠上皮细胞中兴奋性氨基酸转运载体1(EAAC1)、谷氨酰胺载体2(ASCT2)、氨基酸转运载体A2(ATA2)、L型氨基酸转运载体2(LAT2)、阳离子氨基酸转运载体1(CAT1)、b 0,+系统氨基酸转运载体(rBAT)、y+L系统氨基酸转运载体1(y+LAT1)、y+L系统氨基酸转运载体2(y+LAT2)和寡肽转运载体1(PepT1)mRNA相对表达量(P<0.01)。3)0.4 mg/L槲皮素极显著降低猪肠上皮细胞中结节性硬化复合物1(TSC1)mRNA相对表达量(P<0.01);0.8 mg/L槲皮素极显著增加mTOR和核糖体蛋白S6(RPS6)mRNA相对表达量并极显著降低TSC1 mRNA相对表达量(P<0.01);1.6 mg/L槲皮素极显著增加mTOR、真核起始因子4E结合蛋白1(4E-BP1)、真核细胞翻译起始因子4E(eIF4E)、真核细胞翻译起始因子4B(eIF4B)、真核细胞翻译起始因子4A(eIF4A)和RPS6 mRNA相对表达量(P<0.01)。4)0.1和1.6 mg/L槲皮素极显著提高猪肠上皮细胞中mTOR、eIF4E和eIF4A蛋白表达量并极显著降低4E-BP1蛋白表达量(P<0.01)。由此可见,槲皮素可通过调控氨基酸转运载体、小肽转运载体及mTOR信号通路相关基因的表达来促进猪肠上皮细胞对蛋白质的利用。     

In vitro differentiation of canine celiac adipose tissue-derived stromal cells into neuronal cells

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.     

The role of glutamate in central nervous system health and disease--a review

Glutamate is the principal excitatory neurotransmitter in the brain. Knowledge of the glutamatergic synapse has advanced enormously over the last 10 years, primarily through application of cellular electrophysiological and molecular biological techniques to the study of glutamate receptors and transporters. There are three families of ionotropic glutamate receptors with intrinsic cation permeable channels. There are also three groups of metabotropic, G-protein-coupled glutamate receptors that can modify neuronal excitability. There are also two glial glutamate transporters and three neuronal transporters in the brain. Endogenous glutamate may contribute to the brain damage occurring acutely after traumatic brain injury as well as having a role in the excitatory imbalance present in epileptic conditions and contributing to the pathophysiology of hepatic encephalopathy in animals. Understanding the role of glutamate in these neurological diseases may highlight treatment potentials of antagonists to glutamatergic transmission. This paper presents a review of the literature of glutamate and its role in neurological function and disease.     

Impaired expression of excitatory amino acid transporter 2 (EAAT2) and glutamate homeostasis in canine necrotizing meningoencephalitis

To clarify the involvement of excitatory and inhibitory amino acids in canine necrotizing meningoencephalitis (NME), glutamate, aspartate, taurine and gamma-aminobutylic acid (GABA) were determined in the cerebrospinal fluids (CSF) from eight NME cases and ten healthy controls. NME dogs exhibited significantly higher concentrations of glutamate and aspartate than those in controls (p<0.001 and p<0.001, respectively), while there was no difference in taurine or GABA between the two groups. When fetal canine astrocytes were cultured for 24 hr in the presence of NME-CSF, supernatant concentrations of glutamate, aspartate and taurine were significantly elevated. Simultaneously, expression of excitatory amino acid transporter 2 (EAAT2) mRNA was significantly reduced in the astrocytes without change in EAAT1 mRNA. Hence, reduced expression of EAAT2 and impaired glutamate homeostasis may contribute to the pathogenesis of NME.     

Evidence of LAT1 expression in canine caput epididymis

L-type amino acid transporter 1 (LAT1), the first isotype of amino acid transport system L, transports aromatic and branched amino acids pivotal for fundamental cellular activities such cellular growth and proliferation. LAT1 expression was high only in the brain in contrast to its limited distribution and low level of expression in normal tissues. We found potent LAT1 expression in canine caput epididymis by quantitative RT-PCR and Western blotting analysis. Immnuno-histochemical examination revealed observable LAT1 in microvillous epithelial cells.     

Canine Lat1: molecular structure, distribution and its expression in cancer samples

A full-length cDNA sequence of canine L-type amino acid transporter 1 (Lat1) was determined from a canine brain. The sequence was 1828 bp long and was predicted to encode 485 amino acid polypeptides. The deduced amino acid sequence of canine Lat1 showed 93.2% and 91.1% similarities to those of humans and rats, respectively. Northern blot analysis detected Lat1 expression in the cerebellum at 4 kb, and Western blot analysis showed a single band at 40 kDa. RT-PCR analysis revealed a distinct expression of Lat1 in the pancreas and testis in addition to the cerebrum and cerebellum. Notably, Lat1 expression was observed in the tissues of thyroid cancer, melanoma and hemangiopericytoma. Although the cancer samples examined were not enough, Lat1 may serve as a useful biomarker of cancer cells in veterinary clinic.     

谷氨酸和谷氨酰胺转运系统的研究进展

谷氨酸作为幼年动物重要的氨基酸,是肠内能量生成的最大贡献者,它不能由机体自身合成,需额外添加或通过谷氨酸前体物谷氨酰胺转化而成.谷氨酸是谷胱甘肽合成的重要底物,对动物肠道抗氧化剂的提供有重要作用,其转运依靠谷氨酸转运载体完成.因此,本文就谷氨酸和谷氨酰胺转运系统的分类及作用机制做一综述.     

Changes in extracellular neurotransmitters in the cerebrum of familial idiopathic epileptic shetland sheepdogs using an intracerebral microdialysis technique and immunohistochemical study for glutamate metabolism

Intracerebral microdialysis combined with electroencephalographic recordings was performed on 4 dogs of a familial idiopathic epileptic Shetland sheepdog colony to identify the kinds of neurotransmitters responsible for seizure activity. Immunohistochemistry using glutamate (Glu), glutamate transporter (GLT-1 and GLAST), and glutamine synthetase (GS) antibodies was also carried out on the cerebrum of four familial dogs that died of status epilepticus (SE). High values for extracellular levels of Glu and aspartate (ASP) were detected in association with an increased number of spikes and sharp waves during hyperventilation in 3 of 4 the familial epileptic dogs. The values of other amino acids analyzed were not altered in any of the familial epileptic dogs. Immunohistochemically, Glu-positive granules were occasionally found in the perineuronal spaces of the cerebral cortex in 3 of the familial epileptic dogs that died of SE. Immunostains for GLT-1 antibody predominantly decreased in the cerebral cortex and lateral nucleus of the thalamus in all the dogs that died of SE, whereas there were no differences detected in immunolabellings for GLAST and GS antibodies between familial epileptic dogs and controls. These results suggest that an extracellular release of both Glu and Asp may play an important role in the occurrence of seizure activity in this epileptic colony, and that a decreased expression of astrocytic GLT-1 may be related to development of SE.     

小肽转运蛋白(PepT1)基因研究进展

小肽作为蛋白质的主要消化产物 ,在氨基酸消化、吸收和代谢中起着重要作用。小肽与游离氨基酸的吸收是两个相互独立的转运系统 ,与游离氨基酸相比 ,小肽具有吸收速度快、耗能低、不易饱和 ,且各种肽之间转运无竞争性与抑制性等特点。本文主要综述了小肽转运机制的特点和小肽转运蛋白(PepT1)分子生物学方面的研究进展 ,包括PepT1分子结构特点 ,PepT1cDNA的克隆 ,不同动物之间PepT1氨基酸序列的同源性 ,PepT1mRNA在不同动物、不同组织中的分布 ,以及营养水平对PepT1基因表达的影响 ;并就需要进一步深入研究的问题进行了探讨     

Early-age feed restriction affects viability and gene expression of satellite cells isolated from the gastrocnemius muscle of broiler chicks

Background: Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0-14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated. Results: One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T 3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells. Conclusions: These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3 , and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.     

Distribution of Vesicular Glutamate Transporter 2 and Ionotropic Glutamate Receptors in the Auditory Ganglion and Cochlear Nuclei of Pigeons (Columba livia)

Glutamate is a principal excitatory neurotransmitter in the auditory system. Our previous studies revealed localization of glutamate receptor mRNAs in the pigeon cochlear nuclei, suggesting the existence of glutamatergic input from the auditory nerve to the brainstem. This study demonstrated localization of mRNAs for vesicular glutamate transporter 2 (vGluT2) and ionotropic glutamate receptors (AMPA, kainate and NMDA) in the auditory ganglion (AG) and cochlear nuclei (magnocellular, angular and laminar nuclei). VGluT2 mRNA was intensely expressed in AG and intensely or moderately in the cochlear nuclei. The AG and cochlear nuclei showed intense‐to‐moderate mRNA signals for GluA2, GluA3, GluA4, GluK4 and GluN1. These results suggest that the pigeon AG neurons receives glutamatergic input from hair cells and in turn projects to the magnocellular and angular nuclei. Glutamate may play a pivotal role in the excitatory synapse transmission in the peripheral auditory pathway of birds.     

鱼类小肽转运载体PepT1研究进展

小肽作为蛋白质主要的消化产物,与氨基酸的吸收是两个相互独立的转运系统。Peptide Transporter 1(PepT1)是其中一种重要的小肽转运载体。文章就鱼类PepT1的分子特点,PepT1 cDNA的克隆与表达,以及PepT1的转运机制等研究作一综述。     

ABCG2 transporter: therapeutic and physiologic implications in veterinary species

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. While P-glycoprotein, the product of the MDR1 (ABCB1) gene, is the most well-characterized ABC transporter, the pharmacological importance of a related transporter, ABCG2, is starting to be realized in veterinary medicine. Based primarily on human and rodent studies, a number of clinically relevant, structurally and functionally unrelated drugs are substrates for ABCG2. ABCG2 is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain and retinal capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, ABCG2 limits oral absorption of substrate drugs and restricts their distribution to privileged sites such as the brain and retina. ABCG2 is also expressed by tumor cells where it functions to limit the intracellular accumulation of cytotoxic agents, contributing to multidrug resistance. Several ABCG2 polymorphisms have been described in human patients, some of which result in altered drug disposition, increasing susceptibility to adverse drug reactions. Additionally, ABCG2 polymorphisms in humans have been associated with disease states such as gout. Feline ABCG2 has recently been demonstrated to have several amino acid differences at conserved sites compared with 10 other mammalian species. These amino acid differences adversely affect transport function of feline ABCG2 relative to that of human ABCG2. Furthermore, these differences appear to be responsible for fluoroquinolone-induced retinal toxicity in cats and may play a role in acetaminophen toxicity as well. Studies in rodents and sheep have determined that ABCG2 expressed in mammary tissue is responsible for the secretion of many compounds (both therapeutic and toxic) into milk. Finally, data in rodent models suggest that ABCG2 may play an important role in regulating a number of physiologic pathways involved in protecting erythrocytes from oxidative damage.     

地毯草铝响应基因AcABCG1的克隆与表达分析

铝毒抑制植物根系生长,是酸性土壤上限制作物产量的重要因素之一。ABC转运蛋白在金属离子转运和非生物胁迫应答等方面起作用。本研究以地毯草（Axonopus compressus）为材料,克隆了地毯草编码ABC转运蛋白AcABCG1基因,并对其进行了生物信息学和基因表达模式分析。结果表明,AcABCG1基因cDNA全长为2 434 bp,编码811个氨基酸残基,蛋白分子量为91.85 kD。亚细胞定位预测表明AcABCG1定位于细胞质膜上。定量PCR结果发现,金属铝（Al）、镉（Cd）和镧（La）处理均能显著增强AcABCG1基因在地毯草根系中的表达。不同铝浓度和时间处理进一步表明了AcABCG1基因在转录水平上响应铝胁迫。此外,AcABCG1基因在根中的表达量显著高于茎和叶中的表达量,且AcABCG1基因主要在0~1 cm根尖中表达。本研究结果为进一步挖掘AcABCG1基因参与地毯草耐铝毒的生物学功能奠定基础。