Influence of Environmental Salinity on Messenger RNA Levels of Growth Hormone, Prolactin, and Somatolactin in Pituitary of the Channel Catfish (Ictalurus punctatus)

Pituitary growth hormone (GH), prolactin (PRL), and somatolactin (SL) messenger RNA levels in channel catfish (Ictalurus punctatus) were examined under various environmental and physiological conditions. Catfish were sampled following salinity challenge, during the winter (December) and spring or summer (April or July), and at different sizes (15–18 g, 620–664 g, and 956–1134 g). When catfish (956–1134 g) were transferred from freshwater to saline water containing 8 ppt NaCl, their plasma [Na⁺] increased significantly above values in the freshwater control group until they were transferred back to freshwater. Pituitary GH mRNA levels were low for the first 24 hours following transfer to saline water, but thereafter were significantly elevated above control values until the fish were transferred back to freshwater. Pituitary GH mRNA levels were highest in July and lowest in December. Growth hormone mRNA levels were also elevated in the size groups 15–18 g and 956–1134 g in July when compared with December values. Pituitary PRL mRNA levels increased for the first 24 hours following transfer to saline water (956–1134 g), but thereafter were significantly lower than control values until the fish were transferred back to freshwater. Pituitary PRL mRNA levels were highest in April and July and lowest in December, and were also elevated in the size groups 620–664 g and 956–1134 g. Pituitary SL mRNA levels were unaffected in catfish transferred to saline water; however, levels were significantly elevated in catfish of the 956–1134-g size group sampled in April when compared with December. These results suggest the involvement of GH in adaptation to brackish water and of PRL in adaptation to freshwater in the catfish, and seasonal and size-related differences in pituitary GH, PRL, and SL mRNA levels. Received May 17, 2000; accepted October 30, 2000     

生长激素分泌促进剂及构效关系研究进展

生长激素分泌促进剂是一类作用于垂体和下丘脑的具有专一性促生长激素释放作用的寡肽及其类似物.由于其分子质量小、活性高、可口服、作用专一而有可能成为新的生长激素治疗药物.目前已经发展了很多具有此类活性的多种结构的化合物,如肽、环肽、肽醇及非肽类似物等.尽管这类化合物的作用机制尚未完全明确,但已有证据表明存在新的调节生长激素分泌的途径和新的调节因子.     

生长激素信号肽可诱导重组蛋白外分泌表达

重组蛋白质的表达是生物医药开发、基因功能和作用机理研究中关键技术环节.虽然细菌表达体系由于表达量大、经济等而被广泛采用,但由于其不能提供许多蛋白质必需的翻译后修饰如糖基化等,所表达的蛋白又多以不可溶包涵体形式存在,变性复性过程复杂,产率低,因此真核细胞表达体系如CHO、COS等成为活性要求高的蛋白质表达的首选[1].     

生长激素释放肽的合成及促生长活性

用固相多肽合成法合成了生长激素释放肽(GHRP),这是一种含D-型氨基酸的外源性激素,具有促进脑下垂体分泌生长激素功能的人工合成六肽,其氨基酸序列为His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂.观察了使用不同剂量的GHRP对不同日龄小鼠的促生长效应,当使用最佳剂量(1000μg/kg)时,可使25日龄小鼠体重较对照组增加14.8%,同时发现鼠龄越小对GHRP越敏感,当使用剂量高达10mg/kg时仍然安全无毒.     

在肽库中筛选抗磷脂抗体的小肽抑制剂

β２糖蛋白Ⅰ（ｂｅｔａ－２－ｇｌｙｃｏｐｒｏｔｅｉｎ Ⅰ,β２ＧＰＩ）是一分子量为５０ ｋＤ的糖蛋白．从抗磷脂抗体综合症（Ａｎｔｉｐｈｏｓｐｈｏｌｉｐｉｄ ａｎｔｉｂｏｄｉｅｓ ｓｙｎｄｒｏｍ ,ＡＰＳ）患者身上获得的抗体可直接与β２ＧＰＩ作用．以β２ＧＰＩ为目标分子,在噬菌体十五肽库中筛选抗β２糖蛋白Ⅰ抗体的小肽抑制剂．通过四轮亲和性筛选,噬菌体的回收率从１．２６×１０－ ４％ 增加到３．１９×１０－ ２％ ．随机挑取噬菌体克隆测定其与β２ＧＰＩ的结合活性及其对β２ＧＰＩ与自身抗体结合的抑制活性,其中部分噬菌体克隆表现出４０％ 的抑制活性．经ＤＮＡ 序列测定,得到一组相关序列．该结果为研究β２ＧＰＩ的抗原表位奠定了良好的基础．     

猪生长激素及其单克隆抗体的制备与纯化

本文介绍一种简便快捷提纯猪生长激素的方法。通过杂交瘤技术,首次获得抗猪生长激素的单克隆抗体,并讨论和比较从腹水纯化单克隆抗体的各种方法。     

NMDA Receptors in the Medial Zona Incerta Stimulate Luteinizing Hormone and Prolactin Release

1. The aim of the present work is to demonstrate the interaction between the glutamatergic/NMDA and dopaminergic systems in the medial zona incerta on the control of luteinizing hormone and prolactin secretion and the influence of reproductive hormones. 2. Proestrus and ovariectomized rats were primed with estrogen and progesterone to induce high or low levels of luteinizing hormone and prolactin. 2-Amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist, and dopamine were injected in the medial zona incerta. Blood samples were withdrawn every hour between 1,600 and 2,000 hours or 2,200 hours via intracardiac catheter from conscious rats. Additional groups of animals injected with the NMDA receptor antagonist were killed 1 or 4 h after injection. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were measured in different hypothalamic regions. 3. 2-Amino-7-phosphonoheptanoic acid blocked the ovulatory luteinizing hormone surge in proestrus rats. 2-Amino-7-phosphonoheptanoic acid also blocked the increase in luteinizing hormone induced by ovarian hormones in ovariectomized rats, an effect that was partially reversed by dopamine injection. Conversely, the increased release of luteinizing hormone and prolactin induced by dopamine was prevented by 2-amino-7-phosphonoheptanoic acid. We found that the NMDA antagonist injection decreased the dopaminergic activity--as evaluated by the 3,4-dihydroxyphenylacetic acid/dopamine ratio--in the medio basal hypothalamus and increased in the preoptic area. 4. Our results show an stimulatory role of NMDA receptors on the ovulatory luteinizing hormone release and on luteinizing hormone release induced by sexual hormones and demonstrate that the stimulatory effect of dopamine on luteinizing hormone and prolactin is mediated by the NMDA receptors. These results suggest a close interaction between the glutamatergic and dopaminergic incertohypothalamic systems on the control of luteinizing hormone and prolactin release.     

用多抗原肽(MAP)免疫制备单克隆抗体的研究

目前制备单克隆抗体杂交瘤细胞株的传统方法是用纯度很高的天然蛋白作为抗原来免疫动物。此方法虽然成功率很高,但提取和纯化作为抗原用的高纯蛋白确非常困难,在某些情况下甚至是不可能的。为了克服以上不足,近年来发展起用合成多肽作为抗原制备单克隆抗体^[1,2]。这个技术的难点是用合成多肽作为抗原来免疫动物不易获得与天然蛋白呈特异反应的抗体^[3-5]。近年来国外科学家发明了一种称为“多抗原肽”(Multiple antigenic peptide, MAP)物质用作抗原^[6,7]。它是一个由赖氨酸核和多个由同一多肽分子构成的具有免疫原性的大分子。这个大分子的多肽部分组成丁造成动物免疫应答的多个相同抗原决定簇．而赖氨酸核部分只起连接多肽的作用,它本身没有免疫原性。实验证明多抗原肽和佐剂配合使用会引起动物强烈的免疫应答。本文介绍的是从Calpain [EC 3.4.22.17] 28kDa 小亚基上选取的一段氪基酸序列 AAQYNPEPPP-PRTH(氨基酸从73到86)与赖氨酸核偶联形成多抗原肽来制备单克隆抗体。Calpain的水解蛋白活性依附于钙离子浓度。它有两种异构酶：在μmol/L钙离子浓度下被激活的称为“μ-calpain．而在mmol/L钙离子浓度下被激活的称为m-calpain。这两种异构酶都由两个亚基组成。其大亚基的分子量为80kDa,它包括酶的活性区和与钙离子结合区。两种酶的大亚基氨基酸序列各不相同。其小亚基的分子量为28 kDa,这两种酶具有完全相同的小亚基氨基酸序列。有关Calpain的详细资料可参阅文献[8]。     

鲤鱼生长激素基因的改造及其在大肠杆菌中的表达

用聚合酶链式反应（ＰＣＲ）改造了锂鱼生长激素基因,扩增出编码锂鱼生长激素成熟肽的序列,并将此序列克隆到ｐＢｌｕｅｓｃｒｉｐｔ Ⅱ ＫＳ质粒中,序列分析后,亚克隆到大肠杆菌表达载体ｐＢＶ２２０中。经ＳＤＳ－ＰＡＧＥ和薄层扫描,结果表明锂鱼生长激素基因已大肠杆菌中获得表达。锂鱼生长激素成熟肽的分子量为２２０００道尔顿,表达率占细胞总蛋白的１８％以上％。     

利用杆状病毒表达系统表达金鱼生长激素I基因

以不含起始密码ATG的质粒Psxivvi⁺X3为转移载体,将编码金鱼生长激素I的Cdna插入粉纹夜蛾核型多角体病毒(TnNPV)基因组中,构建了形成多角体的重组病毒株TnNPV—SX⁺gfGHl21a。该毒株能利用合成多角体XIV串联启动子,在重组病毒感染的草地贪夜蛾(Spodoptera frugiperda,Sf)昆虫细胞及银纹夜蛾幼虫中表达金鱼生长激素I基因。感染离体细胞及虫俸后的蛋白SDS聚丙烯酰胺凝胶电泳表明．所表达的蛋白分子量为22．5kDa,与理论计算值相符。Westem印迹证实。金鱼生长激素特异蛋白得到表达。     

Predator Avoidance of Transgenic Channel Catfish Containing Salmonid Growth Hormone Genes

Transgenic channel catfish (Ictalurus punctatus) containing salmonid growth hormone genes can grow 33% faster than normal channel catfish under aquaculture conditions. However, before transgenic catfish are released and utilized by the private sector, their genetic impact on the natural environment must be examined. Predator avoidance is one of the major fitness traits determining potential environmental risk. To determine the predator avoidance ability and growth performance of transgenic catfish in a natural habitat, various densities of transgenic and nontransgenic channel catfish were communally stocked in 0.04-ha earthen ponds without supplemental feeding. Largemouth bass (Micropterus salmoides) and green sunfish (Lepomis cyanellus) were stocked as predators. Nontransgenic fry had better predator avoidance than transgenic channel catfish when data were pooled (p < .01). When data were not pooled, nontransgenic catfish had better predator avoidance in six trials and transgenic individuals had better predator avoidance in four trials. There was no difference in predator avoidance in three trials. Overall predator avoidance was also better for nontransgenic individuals (p < .01) when the fish were evaluated as 3.5-g fingerlings, more clearly than as fry, as transgenic individuals were more vulnerable in 3 of 4 trials at this life stage. There was no significant difference in growth performance between transgenic and nontransgenic channel catfish in ponds without supplemental feeding. These findings indicate that transgenic channel catfish could be used for commercial aquaculture without affecting the natural environment. Although transgenic channel catfish may be released to nature by accident, any ecological effect would be unlikely because the increased susceptibility of transgenic channel catfish to predators would most likely decrease or eliminate the transgenic genotype. Received March 8, 1999; accepted June 3, 1999.     

Improved Detection of Human Antibodies to a Plasmodium Antigen Using a Peptide Modified with Aib Residues

A 17-mer sequence was selected as a model to study the influence of modifications of terminal ends both on the conformation of a peptide and on its antigenicity towards naturally developing antibodies. This sequence corresponded to a tandemly repeated motif, found in a long repetitive region, with high helical propensity, of a Plasmodium falciparum liver-stage antigen (LSA-1), immunogenic in man. Our model peptide was synthesized with ionizable or non-ionizable ends, or modified in both extremities by introduction of the helix-promoting residue α-aminoisobutyric acid (Aib). Helical contribution, absent in the 17 amino-acid sequence possessing ionizable ends, was detectable when non-ionizable ends were introduced, and dramatically increased in the Aib-modified analogue. The presence of ionizable ends totally abolished reactivity towards human sera, otherwise detectable with the peptide possessing non-ionizable ends. While modification by Aib residues was neither detrimental nor beneficial to antigenicity in solution, it clearly resulted in an improved sensitivity of the specific antibody detection when used as solid-phase antigen in ELISA. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

猪生长激素cDNA在芽孢杆菌中的表达

利用随机克隆的枯草杆菌启动子-信号序列构建茅孢杆菌分泌载体pUS186。用限制酶将切除了信号序列的猪生长激素cDNA从质粒pLY3-PGH 604切下,亚克隆至pUS186,并在该cDNA的下游接上地衣杆菌α-淀粉酶基因的转录终止子,构建猪生长激素表达质粒pSGH 1864,将此质粒转化蛋白酶双缺陷的枯草杆菌DB104及短小茅孢杆菌289。SDS-聚丙烯酰胺凝胶电泳检出在发酵上清液中多出一条22kD的蛋白带,抗猪生长激素血清免疫印迹法证明这一蛋白带具有免疫活性,表明猪生长激素cDNA已在枯草杆菌及短小茅抱杆菌中表达。     

Body Composition and Tissue Distributions in Growth Hormone Deficient Adults Before and After Growth Hormone Treatment

This study examines short and long-term effects of recombinant human growth hormone (rhGH) on body composition and regional tissue distributions by using a multicompartment technique based on computed tomography. Part I includes nine subjects aged 46 ± 9 years with adult onset GH deficiency who were examined before and in the end of 6 months treatment with rhGH (0.4 U. kg^?1. week^?1) in a double-blind crossover trial. Part II is an ongoing open trial including seven of the males in part I. They were treated with rhGH (0.25 U. kg^?1. week^?1) over an additional period of 24 months. Adipose tissue (AT) was reduced by 4.7 kg (p<0.01) while the muscle plus skin compartment (M) and visceral organs (V) were increased by 2.4 (p<0.05) and 0.7 kg (p<0.01), respectively, over 6 months of treatment with a high rhGH dose. A preferential lipid mobilization occurred in the visceral and subcutaneous trunk depots resulting in a changed AT distribution. Muscles of legs and arms increased while the increase of trunk muscles did not reach significance. The body composition changes were maintained over 2 years additional treatment. The preferential loss in visceral AT was further pronounced while other changes in tissue distributions observed during the first 6 months tended to be reversed on the lower rhGH dosage. It is concluded that growth hormone has profound and discordant effects on AT, M and V and with associated changes in tissue distributions. The beneficial effects on body composition seen in short-term treatment is preserved throughout an additional 24 months period of treatment.     

青鱼生长激素cNDA的克隆与序列分析

从青鱼（Mylopharyngodon piceus）脑下垂体中提取总RNA,采用逆转录PCR法扩增出青鱼生长激素cDNA编码区,克隆到Pucm-T载体上。序列测定表明青鱼生长激素基因的开放阅读框架含有630bp,其中包括22个氨基酸的信号肽和188个氨基酸的成熟肽,青鱼生长激素基因的克隆成功为该基因的功能及应用研究奠定了基础。     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

鲤鱼生长激素在毕赤酵母中的表达

 将编码鲤鱼 (Cyprinuscarpio )生长激素 (GH)成熟肽的cDNA克隆到毕赤酵母 (P .pastoris)胞内表达载体pHIL D2中 ,构建重组表达质粒pHIL D2 GH .转化组氨酸缺陷型酵母GS115,获得表达鲤鱼GH的酵母工程菌 .经甲醇诱导 ,SDS PAGE和Western印迹检测表明 ,鲤鱼GH在酵母中得到表达 ,表达产物在胞内以可溶状态存在 ,具有鲤鱼GH的免疫活性 .用诱导后的酵母投喂罗非鱼 ,实验结果证实所构建的工程菌具有明显的促生长作用     

共表达生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)对动物生长的影响

生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)在序列及功能方面均相似,且同为PACAP/胰高血糖素超家族成员．研究了这二者对生长激素释放的刺激作用,以及对动物生长的影响．构建了3个表达载体,pIRES1- GHRH-PACAP(P-G-P),pIRES1-GHRH(P-G) 及 pIRES1-PACAP(P-P),并转染到CHO细胞中,进行RT-PCR,Dot-ELISA以及Westen-blot检测．此外,给大鼠注射细胞上清表达产物,检测其生物学活性．注射8 h后,注射表达P-G-P上清的大鼠血清中IGF-Ⅰ浓度显著高于其他组(P < 0.05)．用PLGA微球包裹各种质粒,并注射到家兔后肢胫前肌．观察家兔生长情况,并于注射后0,15,30,45天时分别采集家兔血液,检测血液中IGF-Ⅰ浓度．结果显示,三质粒注射组动物体重变化及血液中IGF-Ⅰ浓度均高于对照组．注射后30天时,P-G-P组增重较对照组提高81% (P < 0.01),P-G组比对照组提高15%(P > 0.05),P-P组比对照组高7%(P > 0.05)．另一方面,P-G-P组动物血液中IGF-Ⅰ含量比分别比P-G、P-P及对照组提高16.68% (P > 0.05),17.14%(P > 0.05),50.46%(P < 0.05)．以上结果揭示：给动物注射PLGA微球包裹的共表达GHRH与PACAP质粒,可以增强动物体内生长激素(GH)的分泌,并促进动物生长．通过上述研究发现,肌肉注射PACAP表达质粒可以促进家兔的生长,PACAP和GHRH 共表达可以起到协同作用．这可能为动物的促生长研究提供新的方法．     

Preparation and Characterization of Recombinant Dolphin Fish (Coryphaena hippurus) Growth Hormone

Dolphin fish (Coryphaena hippurus) growth hormone (dfGH) cDNA encoding the mature protein was cloned in a pET11a expression vector and expressed in Escherichia coli BL21 cells upon induction with isopropyl-1-thio-beta-d-galactopyranoside as an insoluble protein. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to homogeneity, as evidenced by SDS-PAGE. Gel filtration on a Superdex column under nondenaturing conditions and amino-terminal analysis showed the purified protein to be monomeric methionyl-dfGH. Binding assays of the (125)I-labeled dfGH to dolphin fish liver microsomal fraction resulted in high specific binding characterized by a K(a) of 0.77 nM(-1) and a B(max) of 285 fmol/mg microsomal fraction protein. The purified dfGH was capable of stimulating proliferation of FDC-P1-B9 cells transfected with rabbit growth hormone (GH) receptor. The maximal effect of dfGH was identical to that of human GH but their respective EC(50) values were 28 nM versus 0.095 nM.