GnRH及其受体在2种真骨鱼消化系统中存在的免疫细胞化学证据

用链霉亲和素—生物素化过氧化物酶复合物(strept avidin biotin—peroxidase complex，SABC)免疫细胞化学方法，使用促性腺激素释放激素(gonadotropin—releasing hormone，GnRH)，促性腺激素释放激素受体(gonadotropinreleasing hormone receptor，GnRHR)2种抗血清对黄颡鱼(Pelteobagrus fulvidraco)和鲇(Silurus asotus)的食道、贲门、胃底、幽门、前肠、中肠、后肠和胰中的免疫活性内分泌细胞进行了定位。结果表明：在鲇的食道、胃、肠、肠固有膜、肠肌间神经丛、胰腺和胰岛中均存在着GnRH和GnRHR免疫活性阳性反应；黄颡鱼消化系统中除了在食道和胰岛中未见GnRH和GnRHR的免疫活性阳性反应外，其他部位均有阳性反应，而且GnRH和GnRHR分泌细胞的分布模式相类似。说明胃肠道中GnRH分泌细胞可能以自分泌或旁分泌方式参与消化功能的调节。本研究首次证实在鱼类的消化系统中存在着GnRH及其受体的免疫活性内分泌细胞，可为GnRH功能的多样性等研究领域提供新的形态学依据。     

介绍效果更好的鱼类催产合剂——高效鱼类催产合剂2号和3号

鱼类促性腺激素(GtH)的分泌和生殖活动已经证明为双重的神经内分泌系统所调节，即促性腺激素释放激素(GnRH)刺激GtH分泌和多巴胺作为促性腺激素释放的抑制因素(GRIF)抑制GnRH的作用以及GtH的自行释放。我们先前对养殖鱼类的研究已证明多巴胺受体的拮抗物(如pimozide，PIM)，排除剂(如reserpine，RES)和多巴胺合成抑制剂都能显著增强LRH-A诱导GtH分泌和排卵的作用。     

鱼类下丘脑—垂体神经分泌系统功能综述

鱼类的生殖是受环境影响的。感觉器官把外界环境的刺激(如温度、光照等)传送到脑,使下丘脑分泌促性腺激素释放激素(GnRH),激发脑垂体分泌促性腺激素作用于性腺并促使性腺分泌性甾类激素,以促使性腺发育成熟及排出精子和卵子。     

高效新型鱼类催产合剂介绍

<正> 近年来对鱼类生殖生理的研究已经证明:(1)鱼类脑垂体促性腺激素(GtH)的分泌活动既受到下丘脑分泌的促性腺激素释放激素(GnRH)的促进,又受到下丘脑产生的促性腺激素释放的抑制因素(GRIF)的抑制。(2)下丘脑神经分泌细胞产生的多巴胺具有GRIF的作用,它既能直接抑制脑垂体GtH细胞的分泌活动,亦能抑制GnRH促进GtH的分泌作用。(3)多巴胺的拮抗物或合成抑制剂能消除多巴胺对鱼脑垂     

促性腺激素释放激素类似物促进鱼类生长激素分泌和生长

采用离体灌流孵育和腹腔注射方法,研究鲑鱼促性腺激素释放激素(GnRH)类似物 sGnRH-A(Arg~6Trp~?Leu~3Pro~?NEt-LHRH)和哺乳类GnRH类似物LHRH-A(Ala~5Pro~?-NEt-LHRH)对鲤鱼脑垂体生长激素(GH)分泌的直接刺激作用以及对草鱼鱼种 GH 分流和生长的促进作用。2分钟脉冲式 sGnRH-A 和 LHRH-A 以剂量依存形式直接刺激离体灌流孵育的鲤鱼脑垂体 GH分泌,且 sGnRH-A 刺激GH 分泌活性显著高于 LHRH-A。腹腔注射两种剂量(0.01μg/g 体重/周和0.001μg/g 体重/周)的 sGnRH-A 或LHRH-A(每周一次,共 6周)都能显著提高草鱼鱼种相对体重和相对体长生长率,并促进草鱼鱼种血清 GH 水平提高。sGnRH-A 和 LHRH-A 促进生长的作用是剂量依存的,且sGnRH-A的促进生长作用显著高于 LHRH-A。     

GnRH基因的研究进展

促性腺激素释放激素(GnRH)是下丘脑分泌的十肽激素,其结构及其生物学功能已成为神经内分泌和生殖生物学研究的热点之一,它是神经、免疫、内分泌三大调节系统相互联系的重要信号分子,对生殖调控具有重要意义。文章阐述了GnRH的来源与分布、分子结构、类型、信号传导、生理功能、表达特性及多态性,同时对近年来国内外关于GnRH及其类似物在动物方面的研究进展进行了综述,为更好地应用这一激素提供参考。     

双螺杆挤压对低值海参多肽提取率及抗氧化活性的影响

海地瓜(Acaudina molpadioides)属低值海参产品,为了提高其使用价值,本研究以海地瓜为原料,利用双螺杆挤压辅助不同浓度亚硫酸钠(Na2SO3)处理,并对挤出物多肽提取率、游离巯基含量和蛋白质溶解度进行测定;利用DA201-C型大孔吸附树脂对挤出物多肽进行分离纯化,并对多肽纯化组分的分子量分布和抗氧化能力进行测定。结果显示,双螺杆挤压辅助3.0% Na2SO3处理组多肽提取率、游离巯基含量和蛋白质溶解度分别为(57.12±0.62)%、(110.32±0.07)%和(28.72±0.13)%,较空白对照组(control check, CK)分别显著提升(7.66±0.35)%、(105.32±0.01)%和(4.91±0.15)% (P<0.05);粗肽经纯化得到纯化组分Ⅰ(1000~3000 u)和纯化组分Ⅱ(<1000 u)。CK组、双螺杆挤压辅助3.0% Na2SO3处理组及其经分离纯化后得到的的纯化组分Ⅰ、Ⅱ海地瓜多肽的DPPH自由基清除率的IC50值分别为25.51、12.72、6.58和9.02 mg/mL,对超氧阴离子自由基清除率的IC50值分别为25.56、13.51、11.87和8.44 mg/mL。纯化组分Ⅰ在浓度为80 μg/mL时,人正常肝细胞(human normal liver cell, LO2)细胞存活率较损伤组最大提高(20.33±0.41)%;纯化组分Ⅱ在浓度为20 μg/mL时,LO2细胞存活率较损伤组最大提高(17.07±1.18)%。综上所述,双螺杆挤压辅助亚硫酸钠处理海地瓜提高了多肽的提取率,并增强了海地瓜多肽抗氧化活性。     

半滑舌鳎(Cynoglossus semilaevis)gnrh2基因克隆、组织分布及卵巢成熟过程中表达分析

为了研究下丘脑神经肽促性腺激素释放激素(Gonadotropin-releasing hormone 2,GnRH2)在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的生理作用,本研究通过RT-PCR及RACE方法获得了半滑舌鳎GnRH2全长cDNA序列;通过实时荧光定量PCR(qPCR)对gnrh2 mRNA的组织分布以及卵巢成熟过程中的时空表达特性进行了分析。结果显示,半滑舌鳎GnRH2全长c DNA序列为538 bp(不包括polyA尾),其中,5'非编码区(Untranslated region,UTR)为154 bp,3'UTR为126 bp,开放阅读框(Open reading frame,ORF)为258 bp,编码85个氨基酸的前体多肽,其分子量及等电点分别为9.69 k Da和8.55。GnRH2前体多肽由信号肽、GnRH2十肽、酶切位点(GKR)以及GnRH相关肽共4部分组成。序列比对分析发现,GnRH2在鱼类中同源性极高,尤其是十肽(QHWSHGWYPG)在所有硬骨鱼类中完全相同。半滑舌鳎GnRH2与鲈形目同源性最高(89.41%–90.59%),其次为鲽形目、鲑形目和鲀形目(78.82%–85.88%),与鲤形目同源性最低(61.18%–71.76%)。gnrh2 mRNA主要在脑中表达,在垂体及其他外周组织中表达量极低。此外,组织学分析显示,半滑舌鳎卵巢发育共分为5个时期(Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ期)。在卵巢成熟过程中,脑gnrh2 mRNA表达量在卵黄生成期(Ⅲ期)显著性增加,达到峰值;随后表达量急剧下降,在成熟期(Ⅴ期)达到最小值;在排卵后期(Ⅵ期)又显著性增加。然而,在卵巢成熟过程中,垂体gnrh2 mRNA表达量在卵黄生成后期(Ⅳ期)显著性降低,随后在成熟期(Ⅴ期)有所增加,但在排卵后期(Ⅵ期)又急剧下降。上述研究结果表明,脑GnRH2可能参与了半滑舌鳎卵巢发育过程。     

利用免疫组化方法初步鉴定雌性三疣梭子蟹视神经节GnRH受体

为了研究促性腺激素释放激素(GnRH)受体在甲壳动物视神经节中的存在及分布情况,采用MaxVisionTM免疫组织化学方法,以兔抗人GnRH受体的多克隆抗体,对不同发育期雌性三疣梭子蟹的视神经节进行了免疫组织化学定位。结果显示,GnRH受体的免疫阳性物质在视神经节的多个部位,视神经层、视外髓、视内髓、视端髓及X器中都有较为广泛的存在,在视外髓、视内髓与X器处的神经分泌细胞中尤为明显。不同发育期的雌性三疣梭子蟹视神经节GnRH受体的免疫阳性分布位置相似,免疫阳性强度存在一定的差异。三疣梭子蟹视神经节存在的GnRH受体免疫阳性物质,为GnRH参与视神经节调节作用提供了形态学依据。     

浅谈生殖激素在奶牛繁殖中的应用

生殖激素是直接作用于生殖活动,并以调节生殖过程为主要生理功能的激素。在生殖过程中具有直接调节母畜的发情、排卵、生殖细胞在生殖道内的运行、胚胎附植、怀孕、分娩、泌乳、以及公畜精子的生产、副性腺分泌等功能。在奶牛繁殖中的运用更是广泛,笔者在临床解决奶牛繁殖障碍病的治疗上运用生殖激素取得了良好的效果。现介绍如下:1生殖激素的分类及其生理功能1.1释放激素1.1.1促性腺激素释放激素(GnRH):它可能促进垂体前叶释放促卵泡素(FSH)及促黄体素(LH)。其常用的类似物质主要有促排2号(LRH—A2),促排3号(LRH—A3),促黄体生成素…     

日本鳗鲡(Anguilla japonica)脾脏小分子抗菌肽的分离纯化

为分离日本鳗鲡脾脏中的抗菌肽,实验采用10 ku和3 ku切向流中空纤维柱对脾脏醋酸粗提物进行分级分离,并结合阳离子交换层析和反相高效液相层析技术对小于10 ku和小于3 ku 2部分样品分别进行分离纯化。结果显示,2种分子量截留纤维柱超滤获得了大于10 ku、小于10 ku和小于3 ku 3部分样品。小于10 ku和小于3 ku蛋白样品抗菌活性较大于10 ku样品强。对小于10 ku样品经阳离子交换层析以及采用pH 9缓冲液进行反相高效液相层析分离纯化,获得一个抗菌肽,质谱分析其分子量为1 391.82 u;对小于3 ku样品经阳离子交换层析并采用pH 9和pH 2两种缓冲液进行反相高效液相层析分离纯化,获得一个抗菌肽,质谱分析其分子量为839.19 u。研究表明,日本鳗鲡脾脏中起抗菌活性的主要成分是一些小分子蛋白。     

Gonadotropin-releasing hormone in two perciform fishes: Micropogonias furnieri and Pagrus pagrus

Gonadotropin-releasing hormone (GnRH) molecular variants were characterized by gradient reverse phase high performance liquid chromatography (RP)-HPLC) from brain extracts of two perciforms with economic importance for Argentina and Uruguay. RP-HPLC fractions were tested in radioimmunoassays (RIAs) with both poly-specific and specific antisera. Both species showed the presence of the same three molecular forms, immunologically and chromatographically indistinguishable from sbGnRH, cGnRH-II and sGnRH. This study supports the hypothesis that their expression is a common pattern in perciforms.     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

Purification and Identification of Antioxidant Peptides from Fermented Fish Sauce (Budu)

This study aimed to isolate antioxidant peptides from Budu extract and identify novel antioxidant peptides using high-performance liquid chromatography (HPLC) and electrospray ionization-time-of-flight mass spectrometry (ESI-TOF MS/MS). An amino acid analyzer was used to analyze the amino acid composition of peptides. Two novel peptides, Lue-Asp-Asp-Pro-Val-Phe-Ile-His (LDDPVFIH) and Val-Ala-Ala-Gly-Arg-Thr-Asp-Ala-Gly-Val-His (VAAGRTDAGVH), were identified. The synthesized peptide of LDDPVFIH showed higher antioxidant activity. The presence of hydrophobic amino acids (Ile and Leu), acidic (Asp) and basic (His) amino acids in the peptide sequences is believed to contribute to the high antioxidant activity of the fermented anchovy fish (Budu) extract. Thus, the two peptides may have potential application as functional foods and could also be used as nutraceutical compounds.     

自由基氧化法制备海带岩藻聚糖硫酸酯的抗凝血活性

以海带(Laminaria japonica)为原料,采用阴离子交换和自由基氧化降解反应制备了不同分子量和硫酸根含量的岩藻聚糖硫酸酯,然后以血浆复钙时间(RT)、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)为指标,研究海带岩藻聚糖硫酸酯抗凝血活性的构效关系.结果表明,采用离子交换,可从海带糖提取物中分离出低硫大分子组分F-A和高硫大分子组分F-B.粗糖经自由基氧化降解,可得到分子量为1.5 kD和8.0 kD左右的低分子组分,实验重复性好,产物分子量集中,其中分子量8.0 kD左右的低分子组分硫酸根含量最高.抗凝血实验表明,岩藻聚糖硫酸酯能显著延长RT、APTT和TT,尤以高硫组分大分子F-B抗凝血效果最好,而对PT影响不大,说明岩藻聚糖硫酸酯对外源凝血系统影响很小,它的抗凝血活性主要是通过内源性凝血途径来实现的.岩藻聚糖硫酸酯分子量对抗凝血活性的影响要比硫酸根含量的影响更大,其抗凝血活性随着分子量的降低而显著降低.对于分子量相近的岩藻聚糖硫酸脂,硫酸根含量越高,则抗凝血活性越高.总之,岩藻聚糖硫酸酯分子量和硫酸根含量越高,其抗凝活性越高.     

Purification of Algal Calcium-Chelating Peptide and Its Physical Chemical Properties

The Schizochytrium protein, extracted from byproduct of Schizochytrium sp. after lipid extraction, was isolated and hydrolyzed. A specific calcium-binding peptide was purified from the hydrolysate through Sephadex G-25 gel filtration chromatography and C18 reversed-phase high-performance liquid chromatography (RP-HPLC). The calcium-binding capacity of the specific peptide reached up to 136.68 ± 4.6 μg/mg. The amino acid sequence was confirmed as Ser-Ser-Val (SSV) with a molecular weight of 291.15 Da by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Fourier transformation infrared spectroscopy showed that the major binding sites included oxygen atom from carbonyl group and nitrogen of amino group or imino group. SSV-Ca chelate was confirmed to be a neutral molecular by Zeta potential analysis, and the calcium ion was surrounded by the functional chelating sites of SSV. In addition, thermogravimetry-differential scanning calorimetry (TG-DSC) and calcium releasing assay revealed that the SSV-Ca chelate exhibited excellent thermal stability and solubility in both acidic and basic conditions, which was in favor for calcium absorption in the gastrointestinal tracts of humans. The findings indicate the by-product of Schizochytrium sp. is a promising source for making peptide-calcium chelate as a dietary functional supplement.     

反相高效液相色谱法分离中华绒螯蟹性腺抑制激素的初步研究

采用反相高效液相色谱层析法(RP-HPLC)分离中华绒螯蟹(Eriocheir sinensis)眼柄视上神经节中的性腺抑制激素,收集在高血糖激素家族区域内出峰的洗脱液组分,得到7组单一的样品组分。通过活体注射的方法,观察和统计每一组分对去眼柄中华绒螯蟹卵母细胞直径大小的影响,以检验每个分离组分的性腺抑制活性。结果发现,其中的第7组分对中华绒螯蟹的卵巢发育具有最强的抑制作用,推测这一组分为中华绒螯蟹的性腺抑制激素。     

Glycoproteins isolated from scallop shells inhibit differentiation of 3T3-L1 preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells. In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.     

Mass spectrometric detection of proopiomelanocortin (POMC)-related peptides following molecular cloning of POMC cDNA in bigeye tuna Thunnus obesus

Several pituitary hormones, including adrenocorticotropin (ACTH), melanotropin (MSH) and β-endorphin, are generated from a common precursor protein, proopiomelanocortin (POMC). In fish, in addition to steroidogenesis of ACTH and melanogenesis of MSH, immunomodulating activity has been found in some POMC-related peptides. To investigate the functions of these peptides in the homologous system, it is necessary to establish a convenient detection method for the peptides. The present study aimed to establish a method for the detection of POMC-related peptides in bigeye tuna Thunnus obesus using a small amount of tissue sample, but not requiring peptide purification. We first determined the nucleotide sequence of tuna POMC cDNA. The cDNA was composed of 1084 base pairs (excluding the poly A tail) that encoded POMC consisting of 222 amino acids. We then fractionated an acid-acetone extract of one pituitary by reverse-phase high performance liquid chromatography (HPLC) and determined the molecular weight of each separated peptide by mass spectrometry. Consequently, we detected eight POMC-related peptides by comparing the values to the deduced amino acid sequence. Thus, the present study enabled the detection of POMC-related peptides from a small amount of tissue without the use of several purification steps.     

大眼金枪鱼头蛋白酶解物1 ku超滤组分的抗氧化活性及其理化性质

研究大眼金枪鱼头蛋白酶解物1 ku超滤组分体外的还原力、自由基清除能力及对衰老小鼠体内抗氧化能力的影响,分析1 ku超滤组分的一般成分、氨基酸组成及分子量分布,为进一步分离纯化金枪鱼头抗氧化肽提供基础。体外结果显示,1 ku超滤组分对羟基自由基、超氧阴离子和DPPH自由基的清除活性随浓度的增加而增强,IC50分别为1.38、0.73与0.93mg/mL,还原力也随浓度的增加而增大,在浓度为12.5 mg/mL时为0.763;体内结果显示,灌胃30 mg/kg的1 ku超滤组分连续42 d,D-半乳糖致衰老小鼠肝组织的超氧化物歧化酶(SOD)活性、肝组织和血清的谷胱甘肽过氧化物酶(GSH-PX)活性显著提高(P<0.05),血清丙二醛(MDA)含量显著降低(P<0.01);理化分析结果显示,1 ku超滤组分(干基计)蛋白质含量为96.40%,脂肪0.11%,灰分4.86%,疏水性氨基酸占氨基酸总量的35.8%,活性组分分子量在1 802~2 519 u和422~922 u。