Molecular cloning and structural analysis of human norepinephrine transporter gene（NETHG）

A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ－aminobutyric acid transporter(hGAT) and a number of other membrane proteins.     

Analysis of expressed receptor-like kinases （RLKs） in soybean

Receptor-like kinases （RLKs） play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags （ESTs） survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.     

Ontogeny of rat chondrocyte proliferation： studies in embryo, adult and osteoarthritic （OA） cartilage

The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic(OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor β1 (TGF-β1) receptors, cyclin dependent kinase (CDK 1)and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-β1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was notexpressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied.Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.     

High IFN－α expression is associated with the induction of experimental autoimmune uveitis （EAU） in Fischer 344 rat

INTRODUCTIONThe CD4 T cells can be subdivided intoTh1 and Th2 subsets based on their secreted cy-tokine profile. Th1 cells characteristical1y secreteInterferonry (IFN--ry), whereas Th2 cells maiuly pro--duce IL--4[1]. IL-12 po1arizes the differentiation ofnaitre, CD4 T cells towards Th1 pathWay, in con-trast IL--4 directs T cell differentiation towards Th2pathWay The broken balance between Th1 and Th2immune responses and predominant Th1 responseare crucial factors in initiation…     

Norepinephrine transporter (NET) knock-out upregulates dopamine and serotonin transporters in the mouse brain

The noradrenaline, serotonin and dopamine transporters are three main transporters, which are the target of the antidepressant drugs. In the present study we demonstrate that the life-long deletion of the noradrenaline transporter (NET) induced up-regulation of two other monoamine transporters, dopamine and serotonin (DAT and SERT, respectively). An increase in the binding of [³H]paroxetine to the SERT and [³H]GBR12935 to the DAT was observed in various brain regions of NET-KO mice, without alterations of mRNA encoding these transporters, as measured by in situ hybridization. This important finding impacts the interpretation of previous data indicating the supersensitizity of NET-KO mice for psychostimulants or stronger effect of citalopram in behavioral tests. While using the NET-KO mice in various psychopharmacological studies is very important, one has to be aware that these mice lack NET from the earliest period of their existence, thus compensatory alterations do take place and have to be considered when it comes to interpretation of the obtained results.     

Agiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats

Recently, the calciumcalmodulindependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and MHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in Ang II treated rats, suggesting that the calciumcalmodulindependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.     

Norepinephrine transporter expression and function in noradrenergic cell differentiations

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-1 (TGF-1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.     

Ectopic discharges trigger sympathetic sprouting in rat dorsal root ganglia following peripheral nerve injury

Experimental backgrounds of ectopic discharges were made by i.p. administrating of 4-aninopyriding (4-AP), a K+ channel blocker, or anisodamine, a muscarinic receptor blocker, in CCI rats, and the sympathetic sprouting in the dorsal root ganglia (DRG) as well as the heat-hyperalgesia was observed. It was demonstrated that the increased ectopic discharges induced by 4-AP promote sympathetic sprouting in the DRG and a greater number of sympathetic basket cells were developed, causing exacerbation of heat-hyperalgesia in CCI rats. On the contrary, the sympathetic sprouting in the DRG and heat-hyperalgesia are evidently diminished after anisodamine injection. Our results suggest that ectopic discharges may be an immediate factor in triggering sympathetic sprouting in DRG following peripheral nerve injury.     

Bilateral sympathetic stellate ganglionectomy attenuates myocardial remodelling and fibrosis in a rat model of chronic volume overload

Reducing sympathetic neurohormone expression is a key therapeutic option in attenuating cardiac remodelling. Present study tested the feasibility of attenuating cardiac remodelling through reducing sympathetic neurohormone level by partial cardiac sympathetic denervation in a rat model of chronic volume overload. Male Sprague‐Dawley rats were randomized into sham group (S, n = 7), aortocaval fistula group (AV, n = 7), and aortocaval fistula with bilateral sympathetic stellate ganglionectomy group (AD, n = 8). After 12 weeks, myocardial protein expression of sympathetic neurohormones, including tyrosine hydroxylase, neuropeptide Y, growth associated protein 43, and protein gene product 9.5, were significantly up‐regulated in AV group compared to S group, and down‐regulated in AD group. Cardiac remodelling was aggravated in AV group compared to S group and attenuated in AD group. The myocardial deposition of extracellular matrix, including collagen I and III, was enhanced in AV group, which was reduced in AD group. Myocardial angiotensin II and aldosterone expressions were significantly up‐regulated in AV group and down‐regulated in AD group. Our results show that bilateral sympathetic stellate ganglionectomy could attenuate cardiac remodelling and fibrosis by down‐regulating sympathetic neurohormones expression in this rat model of chronic volume overload.     

Characterization of 5‘—proximal sequence of mouse GABA transporter gene （GAT—1）

The cDNA molecule encoding the mouse GABA transporter gene(GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library.A positive clone,harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G,was fished out from the library,the 5‘ proximal region and intron 1 were sequenced and analysed,and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences.The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5‘ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay,and Southern-Western blot.The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.     

培养的大脑皮层、海马及交感神经细胞钠、钾和钙离子通道的全细胞记录技术

目的: 建立新生大鼠大脑皮层、海马细胞及交感神经元细胞的培养方法及其钠、钾和钙通道的膜片钳全细胞记录技术.方法: 取出生1～3 d的大鼠大脑皮层、海马及交感神经节,用胰蛋白酶(0.125%)消化组织并分离出神经细胞,种植在涂有多聚赖氨酸的35 mm培养皿中,用高糖的DMEM培养液培养,一周后,镜下可见神经细胞壁光滑、完整,周围有明亮的光润,在高倍倒置显微镜下可见到完整的细胞核及均匀的胞浆,细胞间形成良好的突触连接,可用于细胞膜片钳记录.结果: 用胰蛋白酶消化分离培养的神经细胞,功能状态良好,在膜片钳全细胞记录中,易形成细胞与记录电极间的高阻抗封接,可分别记录到INa、IA、IK和ICa.结论:在神经系统电生理学研究中,此方法可应用于中枢神经系统不同脑区培养以及钠、钾和钙通道电流的记录.     

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.