Pathways of protein secretion in eukaryotes

Protein secretion from cells can take several forms. Secretion is constitutive if proteins are secreted as fast as they are synthesized. In regulated secretion newly synthesized proteins destined for secretion are stored at high concentration in secretory vesicles until the cell receives an appropriate stimulus. When both constitutive and regulated protein secretion can take place in the same cell a mechanism must exist for sorting the correct secretory protein into the correct secretory vesicle. The secretory vesicle must then be delivered to the appropriate region of plasma membrane. Transfection of DNA encoding foreign secretory proteins into regulated secretory cells has provided insight into the specificity of sorting into secretory vesicles.     

CHO表达生长激素释放因子的生物活性测定

在对生长激素释放因子(GRF)基因改造和化学合成，并构建了其真核表达载体pCDNA3-GRF(1-32)的基础上．用Lipofectin将上述载体转染CHO细胞进行瞬时表达，采用体外单层垂体细胞培养的方法对表达的GRF(1-32)进行了体外生物活性测定，即先将垂体用酶进行消化，再将消化细胞培养，形成单层细胞，利用其能合成与释放生长激素的能力来检测GRF的活性。结果表明：表达产物可刺激生长激素释放，并且比对照组提高3．8倍，     

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.     

Vasopressin and neurophysin: high concentrations in monkey hypophyseal portal blood

Vasopressin and its binding protein, neurophysin, were measured by radioimmunoassay in the hypophyseal portal blood of monkeys after cannulation of individual long portal veins. Mean vasopressin concentrations (13,800 picograms per milliliter) in portal blood were more than 300 times as high as those in the systemic circulation (42 picograms per milliliter). Neurophysin concentration was approximately 25 times as high in portal as in systemic blood. By immunoperoxidase techniques, high concentrations of neurophysin were demonstrated around portal capillaries of the median eminence. These studies indicate direct secretion of vasopressin and neurophysin into the portal circulation; the quantities secreted during stress may be sufficient to exert significant effects on secretion of anterior pituitary hormone.     

生长早期泰和鸡与AA鸡血清生长激素水平及生长激素结合蛋白活性的比较

比较泰和鸡和ＡＡ（Ａｒｂｏｒ Ａｃｒｅｓ）肉鸡血清生长激素（ＧＨ）含量和生长激素结合蛋白（ＧＨＢＰ）的结合活性,发现两品种鸡出雏时血清ＧＨ水平均较低,５日龄时ＧＨ均有显著上升（Ｐ＜０．０１）。此后泰和鸡保持上升趋势,在３５日龄ＧＨ达到最高值;而ＡＡ鸡在５日龄后血清ＧＨ水平上升幅度较小,日龄间差异不显著。１日龄和３５日龄时,泰和鸡血清ＧＨ水平显著高于同日龄的ＡＡ肉鸡,与两者体重的差异刚好相反。血液Ｇ     

Receptor-associated resistance to growth hormone-releasing factor in dwarf "little" mice

Anterior pituitaries from the dwarf mouse strain "little" did not release growth hormone or accumulate adenosine 3',5'-monophosphate (cyclic AMP) in response to human and rat growth hormone-releasing factor (GRF). Dibutyryl cyclic AMP, as well as the adenylate cyclase stimulators forskolin and cholera toxin, markedly stimulated growth hormone (GH) release. The basis of the GH deficiency in the little mouse may therefore be a defect in an early stage of GRF-stimulated GH release related either to receptor binding or to the function of the hormone-receptor complex.     

A test of clathrin function in protein secretion and cell growth

Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible. The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion. Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11. Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA. Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains. Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate. Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles.     

侧脑室注射瘦素对大鼠垂体生长激素mRNA表达的影响

为了探讨Leptin(瘦素)对动物生长激素(GH)分泌的影响,以大鼠为试验动物,通过侧脑室注射1 mg Leptin,并采用逆转录多聚酶链反应(RT-PCR)法测定了大鼠垂体GH mRNA表达水平。结果表明,大鼠侧脑室注射Leptin,能显著增加垂体GH mRNA的表达。这一结果证明了Leptin可通过下丘脑调控动物垂体GH的合成与分泌。     

蛋鸡下丘脑中生长抑素神经原发育的研究

应用免疫组织化学技术显示蛋鸡生长发育过程中下丘脑中生长抑素（ＳＳ）免疫阳性神经原的分布。发现蛋鸡在生长早期（１周龄和３周龄）ＳＳ神经原数量较多，６周龄时暂时降低，到９周龄又回升；在生长晚期（６１周龄）呈显著下降趋势。尽管公鸡和母鸡ＳＳ神经原数量没有显著差异，但母鸡的ＳＳ神经原数量从第３周到第６周明显下降，第９周时显著上升，表明ＳＳ神经原的发育具有性别差异。研究结果表明，蛋鸡下丘脑中ＳＳ神经原数量与血浆中ＧＨ水平呈负相关，下丘脑中ＳＳ控制着脑垂体中ＧＨ的分泌。     

导入外源GRF基因质粒对犊牛生长性能的影响

为了研究导入GRF基因质粒对犊牛生长性能的影响,选取36头体重为约102kg西门塔尔杂交牛,随机分成四组每组9头,分别注射含有0mg,3.37mg,6.74mg,10.11mg质粒的5ml生理盐水。在试验过程中,定期进行增重,饲料采食量和生长激素水平的测定。试验结果表明,各处理组均在一定程度上提高了动物的日增重和饲料转化率,其中6.74mg处理组效果最好,日增重比对照组高出17.33%(P<0.01),饲料转化率提高18.05%(P<0.01)。屠宰后进行组织质粒残留检测,未发现有残留。     

日粮不同碘、硒水平对绒山羊绒生长相关激素及酶的影响

为探讨日粮添加不同水平的碘和硒对辽宁绒山羊非产绒期和产绒期血液中褪黑激素（MT）、催乳素（PRL）、类胰岛素生长因子（IGF—I）、生长激素（GH）、甲状腺过氧化物酶（TPO）以及脱碘酶（DIO）的影响,选择36只体况良好、体重相近（（38．0±2．94）kg）的2周岁辽宁绒山羊作为试验动物,采用2×3完全随机试验设计,随机分为6组。分别在基础日粮中添加3个水平的碘（0、2和4mg／kg,DM）和2个水平的硒（0和1mg／kg,DM）进行饲喂。试验结果表明：1）非产绒期,日粮添加碘显著提高了血液中GH和IGF1的浓度,降低了血浆MT的浓度（P〈0．05）,对PRL、TPO和D10浓度没有显著影响（P〉0．05）。添加硒对血液MT、PRL、GH、IGF—I和TPO没有影响,但是显著提高了DIO的浓度（P〈0．05）。2）产绒期,日粮添加碘极显著提高了血液中IGF-I的浓度（P〈0．01）,对MT、PRL、GH、TPO和DIO浓度没有影响。添加硒则显著提高了DIO的浓度（P〈0．05）,对血浆MT、PRL、GH、IGF—1和TP0没有影响。通过2个阶段的比较,综合碘促绒生长作用,推断碘或甲状腺激素促进山羊绒生长的机制可能是通过调控IGF—1分泌途径实现的;硒仅提高了血液中脱碘酶的浓度。     

Thyroid hormone induction of an autocrine growth factor secreted by pituitary tumor cells

Thyroid hormones stimulate the rate of cell division by poorly understood mechanisms. The possibility that thyroid hormones increase cell growth by stimulating secretion of a growth factor was investigated. Thyroid hormones are nearly an absolute requirement for the division of GH4C1 rat pituitary tumor cells plated at low density. Conditioned media from cells grown with or without L-triiodothyronine (T3) were treated with an ion exchange resin to remove T3 and were tested for ability to stimulate the division of GH4C1 cells. Conditioned medium from T3-treated cells was as active as thyroid hormone at promoting GH4C1 cell growth but did not elicit other thyroid hormone responses, induction of growth hormone, and down-regulation of thyrotropin-releasing hormone receptors, as effectively as T3 did. A substance or substances associated with T3-induced growth stimulatory activity migrated at high molecular weight at neutral pH and was different from known growth-promoting hormones induced by T3. The results demonstrate that thyroid hormones stimulate the division of GH4C1 pituitary cells by stimulating the secretion of an autocrine growth factor.     

拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用

【目的】研究拟南芥开花抑制因子TFL1与2个GRFs家族成员GRF4和GRF7之间的互作关系,为进一步解析TFL1抑制植物开花的分子机制奠定基础。【方法】以拟南芥cDNA作为模板,利用基因特异性引物,克隆TFL1、GRF4和GRF7,分别连接入门载体pCR8,经菌落PCR扩增和测序鉴定分别获得这3个基因的入门载体TFL1-pCR8、GRF4-pCR8和GRF7-pCR8。利用LR重组的方法将上述3个入门载体分别与目标载体pGADT7和pGBKT7重组获得酵母双杂交试验载体TFL1-BD、GRF4-AD和GRF7-AD。将TFL1-BD载体分别与GRF4-AD或GRF7-AD载体共同转化酵母感受态细胞,于双缺(-Leu/-Trp)培养基上30℃培养2—3d直至长出酵母克隆,选取合适大小的酵母菌落转移到双缺(-Leu/-Trp)和四缺(-Leu/-Trp/-His/-Ade)缺陷培养基上,通过观察酵母菌落的生长情况判断TFL1与GRFs之间的互作关系。利用LR重组的方法将上述3个入门载体分别与目标载体px-nYFP和px-cYFP重组获得TFL1-nYFP、TFL1-cYFP、GRFs-nYFP、GRFs-cYFP双分子荧光互补试验载体,并分别转化农杆菌感受态细胞。将转化TFL1-nYFP或TFL1-cYFP载体的农杆菌分别与转化GRFs-nYFP或GRFs-cYFP载体的农杆菌共注射烟草叶片,培养48h后在荧光共聚焦显微镜下观察烟草细胞中YFP荧光的表达情况。通过YFP荧光信号的有无来判断TFL1与GRFs之间的互作关系。【结果】成功克隆到拟南芥中的3个基因,分别是534bp的TFL1、888bp的GRF4和798bp的GRF7,并分别获得其入门载体(TFL1-pCR8、GRF4-pCR8和GRF7-pCR8)、酵母双杂交试验载体(TFL1-BD、GRF4-AD和GRF7-AD)和双分子荧光互补试验载体(TFL1-nYFP、TFL1-cYFP、GRFs-nYFP和GRFs-cYFP)。在酵母双杂交试验中,相较于阴性对照组,共同转化TFL1-BD与GRFs载体的酵母菌落在双缺(-Leu/-Trp)和四缺(-Leu/-Trp/-His/-Ade)培养基上都生长较好,结果表明TFL1与GRF4、GRF7在酵母中直接相互作用。在双分子荧光互补试验中,相较于阴性对照组,将转化TFL1-cYFP载体的农杆菌与转化GRFs-nYFP载体的农杆菌共注射烟草细胞之后均在烟草细胞核内产生较强的YFP荧光信号;与此同时,将转化TFL1-nYFP载体的农杆菌与转化GRFs-cYFP载体的农杆菌共注射烟草细胞之后同样在烟草细胞核内产生较强的YFP荧光信号。结果表明,TFL1与GRF4、GRF7在烟草中直接相互作用。【结论】拟南芥开花抑制因子TFL1与GRFs家族的2个成员GRF4和GRF7均直接相互作用。     

Effects of Monoclonal Antibody Against Porcine 40-kDa Adipocyte-Specific Membrane Protein on Endocrine Secretion in Pigs

The present study was to investigate the effect of monoclonal antibody against porcine 40-kDa adipocyte-specific membrane protein on endocrine secretion in pigs, in order to provide the evidence for application of this antibody to reduce excessive fat deposition in pig production. 40 Landrace×Saba pigs were randomly divided into 8 groups： 2 control groups were given saline with 10 mL, respectively, and the 6 treatment groups were given monoclonal antibody against porcine 40-kDa adipocyte-specific membrane protein with 0.1, 0.5, and 1.0 mg ·kg^-1 body weight at 15 or 60 kg body weight, respectively, all treatments were performed by intraperitoneal injection. The results showed that this monoclonal antibody could significantly reduce serum insulin level and increase levels of serum growth hormone （GH）, insulin-like growth factor-1（IGF-1）, triiodothyronine （T3）, and tetraiodothyronine （T4） either at 15 or 60 kg body weight injection. However, more marked effect was observed at 15 kg body weight treatment. Moreover, the dose-dependent effect of this monoclonal antibody on endocrine secretion was also observed. This result revealed that this monoclonal antibody increased secretion of hormones regulating fat lysis and reduced secretion of hormones regulating fat synthesis, suggests the reduction of porcine excessive fat deposition by this monoclonal antibody was carried out through affecting hormones regulating fat metabolism.     

不同性别湖羊生长激素分泌特点的比较

6 头装有颈静脉插管的成年湖羊,雌雄各半,按性别分组,间隔 15 min 连续采取 3 h 的血样,用放射免疫分析法研究了不同性别湖羊生长激素的分泌特点。结果显示成年湖羊的生长激素呈脉冲式分泌;与雌性相比,雄性湖羊生长激素的总体水平高 179．60％(P＜0．01),基线水平高 124．84％(P＜0．01),峰强度高 272．80％(P＜0．01),3 h 峰频率则高一倍(P＜0．01)、而峰持续时间基本不变。以上结果表明湖羊生长激素的分泌有性别的差异,雄性湖羊生长激素分泌的量比雌性湖羊生长激素分泌的量高。     

副溶血性弧菌Ⅲ型分泌系统与Ⅵ型分泌系统的研究进展

副溶血性弧菌(Vibrio parahaemolyticus,VP)是一种常见的革兰氏阴性细菌,主要分布在海洋、海口和河口沉积物中,能够引起人急性肠胃炎、伤口感染和败血症,同时危害水产品养殖业。由该菌引起的食物中毒事件在全世界范围内频繁发生,该菌被认为是微生物性食物中毒的主要病原菌之一。影响副溶血性弧菌致病性的毒力因素包括溶血素、黏附因子、分泌系统等,尤其在分泌系统中,Ⅲ型分泌系统(TypeⅢsecretion systems, T3SS)和Ⅵ型分泌系统(T6SS)与副溶血性弧菌致病性密切相关。T3SS是一种多亚基针状结构,将分泌蛋白通过供体细胞质直接注入受体细胞质;T6SS是一种接触依赖性蛋白分泌装置,将毒素直接注入目标细胞内。通过对Ⅲ型分泌系统和Ⅵ型分泌系统的研究进展进行梳理,从这两种分泌系统的结构出发,并对其功能和调控机制分别进行分析比较,指出了二者在效应蛋白及产生毒素上的不同,找出了二者与副溶血性弧菌定植的关系,以期通过这两者的联系进而深入了解副溶血性弧菌的致病机制,为防治该菌提供科学指导。     

Suppression of prolactin in pigs by Escherichia coli endotoxin

An endotoxin produced by Escherichia coli caused a decrease in prolactin concentrations in the plasma of sows when given at low dosages 2 days postpartum. Five to tenfold increases occurred in the plasma cortisol concentrations. Piglet growth, used as an indicator of milk secretion by the sows, was significantly depressed after the endotoxin administration. Some cases of lactation failure in the periparturient sow may thus be due to endotoxins suppressing prolactin concentrations. This appears to be the first report of a bacterial endotoxin having an effect on prolactin in any species.     

Release of multiple hormones by a direct action of interleukin-1 on pituitary cells

Exposure to bacterial endotoxins has long been known to stimulate the release of anterior pituitary hormones; administration of endotoxin was at one time a common clinical test of anterior pituitary function. Endotoxin is a potent stimulus for production of the endogenous pyrogenic protein, interleukin-1 (IL-1), by macrophages and monocytes. The possibility that IL-1 has a direct effect on the secretion of hormones by rat pituitary cells in a monolayer culture was investigated. Recombinant human IL-1 beta stimulated the secretion of adrenocorticotropic hormone, luteinizing hormone, growth hormone, and thyroid-stimulating hormone. Increased hormone secretion into culture supernatants was found with IL-1 concentrations ranging from 10(-9) M to 10(-12) M. Prolactin secretion by the monolayers was inhibited by similar doses. These concentrations of IL-1 are within the range reported for IL-1 in serum, suggesting that IL-1 generated peripherally by mononuclear immune cells may act directly on anterior pituitary cells to modulate hormone secretion in vivo. Incubation of IL-1 solutions with antibody to IL-1 neutralized these actions. These pituitary effects of IL-1 suggest that this monokine may be an important regulator of the metabolic adaptations to infectious stressors.     

半胱胺对湖羊生长激素分泌特点的影响半胱胺对湖羊生长激素分泌特点的影响

对4只装有颈静脉导管的成年湖羊(自身对照)按60mg／kg     

Human prolactin: 24-hour pattern with increased release during sleep

Human prolactin was measured in plasma by radioimmunoassay at 20 minute intervals for a 24-hour period in each of six normal adults, whose sleep-wake cycles were monitored polygraphically. A marked diurnal variation in plasma concentrations was demonstrated, with highest values during sleep; periods of episodic release occurred throughout the 24 hours.