Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.     

红砂促分裂原活化蛋白激酶MAPK基因cDNA片段克隆及序列分析

研究了参与非生物胁迫的促分裂原活化蛋白激酶MAPK基因在红砂抗旱中的作用.通过实验克隆得到了红砂MAPK基因550 bp的cDNA片段,与已知的植物MAPK基因的相应片段表现出较高的同源性(最高达80.5%).MAPK基因在红砂叶片和茎中无组织特异性表达,且随着干旱胁迫程度的加剧其表达量增加,说明MAPK基因在红砂抗旱中起重要作用.     

Molecular cloning and characterization of the rat NMDA receptor.

A complementary DNA encoding the rat NMDA receptor has been cloned and characterized. The single protein encoded by the cDNA forms a receptor-channel complex that has electrophysiological and pharmacological properties characteristic of the NMDA receptor. This protein has a significant sequence similarity to the AMPA/kainate receptors and contains four putative transmembrane segments following a large extracellular domain. The NMDA receptor messenger RNA is expressed in neuronal cells throughout the brain regions, particularly in the hippocampus, cerebral cortex and cerebellum.     

三维银配位聚合物晶体结构及性质研究

目的基于多功能配体2,2’-联吡啶-3,3’-二甲酸合成其银的配位聚合物{[Ag4(L)2.(H2O)].3(H2O)}n(1)(其中L=2,2’-联吡啶-3,3’-二甲酸),并对其性质进行研究。方法使用水热法合成该配位聚合物,采用X-射线单晶衍射测定其结构,并对其进行了红外光谱及元素分析。结果结构研究发现配合物1为3D金属有机框架结构,属正交晶系,空间群为Pbcn,a=13.889(4),b=13.470(4),c=13.852(4),α=90°,β=90°,γ=90°。结论配合物的每个不对称结构单元中包含2个晶体学独立的Ag(I)原子,1个配体L,0.5个配位水分子和1.5个游离水分子。每个配体通过桥联了6个不同的Ag(I)原子构筑了一例具有三维框架结构的配位聚合物。     

Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene

Phosphoenolpyruvate carboxylase (PEPC) plays diverse physiological functions during plant development. In this study, a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton (Gossypium hirsutum cv. zhongmian 35) by RACE-PCR. The cloned cDNA of GhPEPC2 is 3,364 bp in length, and has an open reading frame of 2,913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs. Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome. The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cotton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers. In addition, the recombinant GhPEPC2 has been purified by expressing it in E. coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C₃ PEPC with a higher K_m (83.6 μM) and lower V_max (8.0 μmol min^-1 mg^-1) compared with the C₃ PEPCs previously reported.     

Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene

Phosphoenolpyruvate carboxylase （PEPC） plays diverse physiological functions during plant development. In this study, a new phos- phoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton （Gossypium hirsutum cv, zhongrnian 35） by RACE-PCR, The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110,6 kD and pI of 5,56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs, Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome, The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cot- ton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers, In addition, the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km （83,6 p.M） and lower Vmax （8,0 p.mol min^-1 mg^-1） compared with the C3 PEPCs previously reported.     

BDNF controls dopamine D3 receptor expression and triggers behavioural sensitization

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, is a polypeptidic factor initially regarded to be responsible for neuron proliferation, differentiation and survival, through its uptake at nerve terminals and retrograde transport to the cell body. A more diverse role for BDNF has emerged progressively from observations showing that it is also transported anterogradely, is released on neuron depolarization, and triggers rapid intracellular signals and action potentials in central neurons. Here we report that BDNF elicits long-term neuronal adaptations by controlling the responsiveness of its target neurons to the important neurotransmitter, dopamine. Using lesions and gene-targeted mice lacking BDNF, we show that BDNF from dopamine neurons is responsible for inducing normal expression of the dopamine D3 receptor in nucleus accumbens both during development and in adulthood. BDNF from corticostriatal neurons also induces behavioural sensitization, by triggering overexpression of the D3 receptor in striatum of hemiparkinsonian rats. Our results suggest that BDNF may be an important determinant of pathophysiological conditions such as drug addiction, schizophrenia or Parkinson's disease, in which D3 receptor expression is abnormal.     

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.     

Selective inhibition of cocaine-seeking behaviour by a partial dopamine D3 receptor agonist.

Environmental stimuli that are reliably associated with the effects of many abused drugs, especially stimulants such as cocaine, can produce craving and relapse in abstinent human substance abusers. In animals, such cues can induce and maintain drug-seeking behaviour and also reinstate drug-seeking after extinction. Reducing the motivational effects of drug-related cues might therefore be useful in the treatment of addiction. Converging pharmacological, human post-mortem and genetic studies implicate the dopamine D3 receptor in drug addiction. Here we have designed BP 897, the first D3-receptor-selective agonist, as assessed in vitro with recombinant receptors and in vivo with mice bearing disrupted D3-receptor genes. BP 897 is a partial agonist in vitro and acts in vivo as either an agonist or an antagonist. We show that BP 897 inhibits cocaine-seeking behaviour that depends upon the presentation of drug-associated cues, without having any intrinsic, primary rewarding effects. Our data indicate that compounds like BP 897 could be used for reducing the drug craving and vulnerability to relapse that are elicited by drug-associated environmental stimuli.     

Molecular cloning of cDNA for murine interleukin-3

The cDNA sequence for murine interleukin-3, one of the colony stimulating factors that regulate haematopoiesis, codes for a polypeptide of 166 amino acids including a putative signal peptide. The predicted amino acid sequence indicates that formation of mature interleukin-3 involves proteolytic removal of not only the signal peptide but additional amino-terminal amino acids.     

Molecular cloning of the receptor for human antidiuretic hormone.

Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.     

Molecular cloning and characterization of a cotton phosphoen01pyruVate carboxylase gene

Phosphoenolpyruvate carboxylase(PEPC)plays diverse physiological functions during plant development.In this study,a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton(Gossypium hirsutum CV.zhongmian 35)by RACE-PCR.The cloned eDNA of GhPEPC2 is 3364 bp in length,and has an open reading frame of 2913 bp,encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56.The deduced amino acid sequence Of GhPEPC2 shares high similarity with other reported plant PEPCs.Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome.The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cotton and accumulated highly in roots.flowers and embryos but relatively low in stems and fibers.In addition.the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated.The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km(83.6 μM)and lower Vmax(8.0 μmol min-1mg-1)compared with the C3 PEPCs previously reported.     

Cloning and expression of a rat D2 dopamine receptor cDNA

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological and biochemical characteristics. The D2 dopamine receptor has been implicated in the pathophysiology and treatment of movement disorders, schizophrenia and drug addiction. The D2 dopamine receptor interacts with guanine nucleotide-binding proteins to induce second messenger systems. Other members of the family of receptors that are coupled to G proteins share a significant similarity in primary amino-acid sequence and exhibit an archetypical topology predicted to consist of seven putative transmembrane domains. We have taken advantage of the expected nucleotide sequence similarities among members of this gene family to isolate genes coding for new receptors. Using the hamster beta 2-adrenergic receptor gene as a hybridization probe we have isolated related genes including a cDNA encoding the rat D2 dopamine receptor. This receptor has been characterized on the basis of three criteria: the deduced amino-acid sequence which reveals that it is a member of the family of G-protein-coupled receptors; the tissue distribution of the mRNA which parallels that of the D2 dopamine receptor; and the pharmacological profile of mouse fibroblast cells transfected with the cDNA.     

Molecular cloning and characterization of an insulin-regulatable glucose transporter

A major mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.     

Molecular cloning and expression of cDNAs for the human interleukin-2 receptor

We have purified the human T-cell growth factor (interleukin-2) receptor and have cloned, sequenced and expressed cDNAs corresponding to this receptor. We identify one gene, but two interleukin-2 receptor mRNAs which differ in their polyadenylation signals. We have isolated an additional cDNA that may correspond to an alternatively spliced mRNA that lacks a 216 base segment and appears to encode an altered membrane protein which cannot bind interleukin-2.     

Molecular cloning and expression of a rat V1a arginine vasopressin receptor.

The neurohypophyseal hormone arginine vasopressin has diverse actions, including the inhibition of diuresis, contraction of smooth muscle, stimulation of liver glycogenolysis and modulation of adrenocorticotropic hormone release from the pituitary. Arginine vasopressin receptors are G protein-coupled and have been divided into at least three types; the V1a (vascular/hepatic) and V1b (anterior pituitary) receptors which act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+, and the V2 (kidney) receptor which is coupled to adenylate cyclase. We report here the cloning of a complementary DNA encoding the hepatic V1a arginine vasopressin receptor. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA corresponding to the cDNA is distributed in rat tissues known to contain V1a receptors.     

Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.     

猪GPR84基因克隆和组织表达图谱分析

本研究以长白猪(Landrace)小肠cDNA为模板,克隆游离脂肪酸家族受体GPR84基因cDNA全长.GPR84基因cDNA全长1243bp,编码396个氨基酸的前体蛋白,与人、大鼠、小鼠、牛等哺乳动物的GPR84氨基酸序列一致性分别为90.2%、83.8%、82.8%、89.4%;结构分析表明猪GPR84含有保守的七次跨膜结构域.采用RT-PCR方法,对家猪GPR84基因进行组织表达分析.结果显示:GPR84在家猪各组织中均有表达.其在下丘脑,肺,肝脏及肠末端分区等组织中表达量较高.     

三维海胆状薄水铝石(AlOOH)的合成与表征

在甲醇-水溶液体系中,在无模板剂的情况下,使用无水三氯化铝作为原料,水热合成了由纳米棒自组装成的三维海胆状薄水铝石（AlOOH）超结构.采用XRD、SEM和TEM对其进行了表征,研究结果表明：该海胆超结构是由直径60-80 nm、长度2μm左右的纳米棒自组装而成,形貌规则,分散均匀,其直径为6-8μm,在其形成过程中,定向附着机制起到关键性的作用,推断了纳米棒自组装海胆状超结构的形成机理.