猪胰脏中胰岛素拮抗肽的分离纯化及其性质的初步研究

从猪胰脏的酸醇提取液中纯化了一个新的活性多肽——胰岛素拮抗肽,它在整体和细胞水平上对胰岛素都有明显的拮抗作用。猪胰脏的酸醇提取液经CM-52、BioGel P-6、DEAE-52及RP-HPLC纯化后,可得到纯的胰岛素拮抗肽。它能剂量相关地抑制胰岛素在离体大鼠脂肪细胞中的促脂合成活性,抑制50％胰岛素活性时所需的胰岛素拮抗肽为2.0×10~(-10)mol/L与被拮抗的胰岛素剂量在同一水平上。该肽含有较多的碱性氨基酸,分子量的3 000,其N-末端是封闭的。胰岛素拮抗肽的上述理化特征及其对胰岛素的拮抗活性均不同于目前已知的胰脏活性多肽。它对脂肪细胞中胰岛素的拮抗作用可能具有重要的生理意义。     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

促胰岛素释放肽和GLP-1类似物研究进展

近年来,从细菌、真菌等低等生物和爬行类、哺乳类等高等动物的体内,都发现存在着结构和功能相关、相似的促胰岛素释放肽或GLP-1类似物.目前国内外研究都在密切关注胰高血糖素样肽-1(glucagonl-ikepeptide-1,G LP-1)和G LP-1类似物等胰高血糖素家族肽,对其进行基因工程高效表达或通过组合化学方法修饰、改造,从而设计治疗Ⅱ型糖尿病的多肽类药物.但是,从天然生物体内,尤其是最近从两栖类动物皮肤分泌液中和响尾蛇毒素中发现了大量能稳定促进胰岛素释放的生物活性肽,却还没有受到足够的重视,它们将很可能为筛选和开发出安全、高效、半衰期长的治疗Ⅱ型糖尿病新药物提供全新的思路和广阔的前景.     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

蛙皮抗菌肽促胰岛素分泌功能研究进展

随着对蛙皮抗菌肽功能研究的不断深入,陆续发现部分肽具有促胰岛素分泌活性,该活性对于2型糖尿病治疗具有较好的应用前景。蛙皮抗菌肽即可以通过克服注射胰岛素产生的低血糖反应,又能改善2型糖尿病胰岛素抵抗的问题,这使其有希望成为安全、高效治疗2型糖尿病药物的新药物。本文综述了具有促胰岛素分泌功能的蛙皮抗菌肽的序列特征和工作机制的研究进展,为进一步开展相关研究提供参考。     

发现血管紧张肽的新功能——抗阿片作用

自从在脑内发现阿片肽以来,有人曾在脑内寻找是否存在着内源性的抗阿片物质。目前知道,促黑激素释放抑制因子、ACTH、β-内啡肽片断及八肽胆囊收缩素等均具有对抗阿片物质的作用。但实际上,这些肽都不是以抗阿片物质的形式真正从脑组织中分离提取出来。本文作者用100只牛脑(40kg),先经丙酮提取得到7.5kg干粉。然后,经分子筛、离子交换以及高压液相层析等步骤多次分离纯化,最终得到两个活性峰A和B。活性峰B已纯化为单一肽,并经氨基酸成分分析与[缬~5]-血管紧张肽Ⅰ(十肽)一致。在高压液相中该肽与合成的[缬~5]-血管紧张肽Ⅰ具有相同的保留时间(Rt)。A峰尚未完全纯化,但在四种不同流洗系统高压液相上的Rt与合成的[缬~5]-血管紧张肽Ⅱ     

鳜胰岛素样生长因子-I cDNA全长克隆及组织表达分析

采用RT-PCR、cDNA末端快速扩增法(RACE)等技术克隆了鳜(Siniperca chuatsi)肝组织胰岛素样生长因子-I(IGF-I)cDNA全长序列.结果表明,鳜IGF-I cDNA全长1 784 bp,包括5'端非翻译区233bp,3'端非翻译区990 bp和开放阅读框561 bp,共编码186个氨基酸;前肽由信号肽、成熟肽、E肽三部分组成,其中,信号肽44个氨基酸,成熟肽68个氨基酸,E肽74个氨基酸;成熟肽由B、C、A、D 4个区域组成,E肽分析表明,鳜IGF-I属Ea-4型.鳜IGF-I氨基酸序列与其他脊椎动物IGF-I氨基酸序列相似度达81%～99%,表明IGF-I在脊椎动物进化中较为保守.运用实时荧光定量RT-PCR技术检测了鳜IGF-I在成鱼不同组织中的表达水平,其中,肝中表达量最高,肾、脑、后肠、皮肤、性腺表达量次之,胃、脾、前肠、鳃、心、肌肉表达量较低.本研究结果为该基因的时序表达、生长调控等研究奠定了分子基础.     

横断山区四种湍蛙的细胞遗传学研究

通过染色体组型分析,C带(BSG技术)分析及一种简便的Ag-NORs带分析,对四川湍蛙、理县湍蛙、棕点湍蛙和棘皮湍蛙的种间关系、染色体的演化及其性染色体等问题进行了初步探讨。结果表明:(1)四川湍蛙、理县湍蛙和棕点湍蛙之间的亲缘关系较近,而它们与棘皮湍蛙的亲缘关系较远;(2)在近缘种的分化中,染色体结构异染色质的变化和臂间倒位是重要的因素之一,这在小型染色体上表现得尤为突出;(三)四川湍蛙具有在形态上分化很明显的性染色体。C带分析表明,此性染色体主要由常染色质构成,但在其Y染色体的长臂上存在明显的中间C带,推测尚处于性染色体分化的初期阶段。     

[A6-Ala,A11-Ala]-人胰岛素突变体的构建及其生物活性

利用 PCR技术 ,将胰岛素原基因中 A6和 A1 1的 Cys序列突变成 Ala序列 ,以去除 A链的链内二硫键 .突变基因连接到质粒 p BV2 2 0 ,构建了表达质粒 p JG40 3,并且在大肠肝菌中得到表达 .经过 Sephadex G- 50层析可得到纯化的突变人胰岛素原 ( Mut- HPI- Ala) .纯化产物用胰蛋白酶和羧肽酶处理 ,并经 Resource Q阴离子交换柱层析可进一步获得纯化的突变人胰岛素 ( Mut- HI- Ala) .Native- PAGE分析表明 Mut- HI- Ala的结构松散 .Mut- HPI- Ala的放射免疫活性和胰岛素受体结合活性是非突变人胰岛素原 ( Met- HPI)的 4.6%和 2 .4% .Mut- HI- Ala的放射免疫活性和胰岛素受体结合活性是非突变人胰岛素 ( Met- HI)的 4.3%和 4.6% .实验结果表明 ,胰岛素 A链内的二硫键对胰岛素的生物活性起着重要的作用     

重组单链胰岛素的连接肽与其生物功能的关系

为研究重组单链胰岛素的生物活性与连接肽之间的关系,用基因定点突变的方法分别以二肽A-K,七肽A-A-A-A-A-A-K和十二肽A-A-A-A-A-A-A-A-A-A-A-K连接胰岛素的B30和A1,得到3个单链胰岛素分子PIP,[A]₅PIP和[A]₁₀PIP.它们的受体结合能力分别为胰岛素的0.14%, 14.3% 和11.1%, 体内生物活性与受体结合能力一致,而它们的促生长活性分别为胰岛素的17%,116.3%和38%.结果表明:(ⅰ)单链胰岛素也具有胰岛素的促代谢和促生长功能;(ⅱ)单链胰岛素与胰岛素受体的结合能力与连接肽的长度和氨基酸组成密切相关, 其受体结合能力随连接肽的改变,可由无到100%,进一步说明胰岛素与其受体结合时,B链C端远离A链N端是必需的;(ⅲ)单链胰岛素的促细胞生长能力也与连接肽长度和组成密切相关,且比其自身的促代谢能力强.     

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36-43, 46-54 and 57-63 showed the significant 2-8-fold increase in insulin-releasing activity (P<0.001) compared with 5.6mM of glucose alone. A pool of fractions 36-43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5-3-fold increase in insulin release (P<0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI.... These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.     

Partial purification of an insulin-releasing activity in human serum

An activity that enhances insulin release from perifused rat pancreatic islets has recently been isolated from human serum fractions (molecular weight 1,000–5,000 daltons). To characterize this activity we have studied the insulin-releasing effect of serum subfractions from obese and non-obese children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum insulin-releasing activity eluted in the HPLC system at 12–13 minutes, which corresponded to the retention time of the tridecapeptide insulin-glucagon liberin isolated from bovine hypothalamus. Insulin-releasing activity was found in serum subfractions from both obese and normal-weight children. The relative insulin-releasing potency of the active subfractions was higher than that of the original total serum fractions, indicating the presence of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral glucose loading increased the relative insulin-releasing activity in the HPLC subfractions from obese children. This study suggests that the insulin secretagogue in human serum might be identical to hypothalamic insulin-glucagon liberin as these substances behave similarly on reversed-phase HPLC and have parallel insulin-releasing properties.     

Major contributions of comparative endocrinology to the development and exploitation of the incretin concept

An incretin is a factor released by the gut in response to nutrients that facilitates uptake of glucose by peripheral tissues. The incretin concept predates the discovery of insulin but it is now clear that incretins act by stimulating secretion of this hormone. As glucagon has insulin-releasing activity, it was speculated that intestinal glucagon-like immunoreactivity (enteroglucagon) was involved in the incretin effect but it was an achievement in the field of comparative endocrinology that led to the demonstration that the preproglucagon gene encodes the most potent incretin in the human. Characterization of cloned cDNAs encoding two preproglucagons from the Brockmann body of the anglerfish Lophius americanus demonstrated that the glucagon sequence is flanked by a 34 amino-acid-residue sequence with appreciable structural similarity to glucagon that was termed glucagon-like peptide (GLP). A 36 amino-acid-residue ortholog of anglerfish GLP was subsequently identified in human preproglucagon but this peptide had only weak insulin-releasing activity. However, alignment of GLP sequences from human and teleost fish showed that the human ortholog is extended from its N-terminus by a hexapeptide. Removal of this extension by an endogenous protease generates GLP-1-(7-36)amide, the potent and effective form of the incretin. More recently, comparative endocrinology has contributed to the exploitation of incretins as antidiabetic drugs. Exendin-4, a GLP-1 receptor agonist first isolated from the venom of the Gila monster Heloderma suspectum, is a clinically valuable, long-acting incretin and the skins of several species of frogs synthesize potent insulin-releasing peptides with therapeutic potential.     

Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent.

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography (HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P < 0.001) compared with 5.6 mm glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin-releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin-releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.     

Jejunal factor stimulating insulin release in the isolated perfused canine pancreas and jejunum.

In the present study using an isolated perfused preparation of canine jejunum and pancreas, an insulin-releasing factor was found in the venous effluent of the jejunum. Insulin secretion by the pancreas rose twofold after 10% glucose was infused in the lumen of the jejunum and remained at a high level even after the stimulus was discontinued. No modification of the exocrine pancreatic secretion occurred during the insulin release, and therefore it seems unlikely that gastrin, secretin or cholecystokinin-pancreozymin were released by the jejunal mucosa. In control experiments the values of hyperglycaemia observed previously and intraluminal hyperosmolarity were tested: at these levels, they did not affect insulin secretion. The nature of this intestinal insulin-releasing factor remains unknown however, but may be identifiable when intestinal hormones in blood can be assayed reliably.     

Isolation and Characterisation of Insulin-Releasing Compounds from <Emphasis Type="Italic">Pseudechis australis</Emphasis> and <Emphasis Type="Italic">Pseudechis butleri</Emphasis> Venom

Crude venom from two elapid snakes Pseudechis australis and Pseudechis butleri was fractionated by gel filtration chromatography and selected fractions screened for in vitro insulin-releasing activity using clonal pancreatic BRIN-BD11 cells. Following acute 20-min incubation at 5.6 mM glucose, 9 fractions exhibited significant (P < 0.001) insulin-releasing activity. Structural characterisation of active fractions was achieved primarily using MALDI–TOF MS and N-terminal Edman degradation sequencing. The partial N-terminal sequences are reported for a total of 7 venom components. Their homology to existing sequences as determined using BLAST searching uncovered the main insulin-releasing families as being phospholipases A₂ and short α-neurotoxins. A number of sequences are reported for the first time from P. butleri venom which is much less studied than the related P. australis.     

Isolation and structural characterisation of a novel 13-amino acid insulin-releasing peptide from the skin secretion of Agalychnis calcarifer

We describe the isolation and characterisation of an insulinotropic peptide from the skin secretions of Agalychnis calcarifer frogs. Peptides in crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC, yielding fractions in two zones with insulin-releasing activity ( p <0.001). The peaks showing greatest in vitro insulin-releasing activity were subsequently purified to homogeneity, revealing a novel insulinotropic 13-amino-acid (1653.2 Da) peptide with the primary structure RRKPLFPFIPRPK [corrected] (RK-13). A database search for RK-13 showed 53.8% similarity with the N-terminal region of proline-arginine-rich antimicrobial peptide (PR-39). Synthetic RK-13 stimulated insulin release in a dose-dependent, glucose-sensitive manner, exerting its effects through a cyclic AMP-protein kinase A pathway independent of pertussis toxin-sensitive G proteins. Unlike PR-39, RK-13 lacks antimicrobial effects on the growth of yeast, and Gram-positive and Gram-negative bacteria. Our data indicate that skin secretions of Agalychnis calcarifer frogs contain insulin-releasing peptides, including RK-13, which merit further investigation as insulin secretagogues.     

Evaluation of natural substances from Evolvulus alsinoides L. with the purpose of determining their antioxidant potency

In recent years, great attention has been given to the search for natural compounds or extracts with the purpose of medical use. Evolvulus alsinoides L. (Convolvulaceae) is a plant used in traditional medicine of East Asia in many indications and has known nootropic and anti-inflammatory activity. However, the bioactive constituents have been described poorly in the literature. Four substances isolated from the ethanol extract of E. alsinoides by means of polyamide and Silica-gel chromatography are reported here. Their molecular structures were determined using NMR analyses. There were identified as scopoletin, umbelliferone, scopolin and 2-methyl-1,2,3,4-butanetetrol. The quantity of these substances was determined using HPLC-UV and GC-FID detection. Antioxidant activity of the isolated substances was measured by DPPH assay using the SIA method. Antioxidant activity and total phenolic content of the prepared fractions are also described. The prepared fractions and isolated substances did not exhibit any significant activity in DPPH test.     

Hybridization of non-sulfonylurea insulin secretagogue and thiazolidinedione-derived insulin sensitizer

Hybrid compounds of non-sulfonylurea insulinotropic agents and thiazolidinedione-derived insulin-sensitizing agents were designed and synthesized. The benzylidenesuccinic acid derivative 24 was equal both to nateglinide in potency of insulin-releasing activity and to pioglitazone in insulin-sensitizing activity.     

Skin secretion of the toad Bombina variegata contains multiple insulin-releasing peptides including bombesin and entirely novel insulinotropic structures

Skin secretions of the toad Bombina variegata were evaluated for the isolation and characterisation of insulinotropic peptides. Crude secretions obtained from young adult toads by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 44 peaks. In acute incubations with glucose-responsive BRIN-BD11 cells, peaks 21, 22, 23, 24 and 25 showed a 1.5-3.5-fold increase in insulin release compared with 5.6 mM glucose alone (p<0.001; n=3). Structural analyses of the purified insulin-releasing peaks were performed by automated Edman degradation and mass spectrometry. Peptides represented by peaks 21, 22 and 23 had molecular masses of 1641.7 Da, 1662.6 Da and 1619.8 Da respectively. These peptides were unblocked by removal of pyroglutamic acid from the N-terminus prior to Edman degradation, revealing lengths of 14 amino acids. Peak 21 yielded a primary structure of Pyr-QRLGHQWAVGHLM, which a data base search revealed as an analogue of bombesin (His6 bombesin), while peak 23 was an exact match of bombesin (Pyr-QRLGNQWAVGHLM) originally isolated from Bombina bombina. Peak 22 indicated a primary structure of Pyr-DSFGNQWARGHFM (72% homology with bombesin). Peaks 24 and 25 revealed entirely novel insulinotropic peptides with molecular masses and primary structures of 1650.5 Da and 2300.0 Da and GKPFYPPPIYPEDM (GM-14) and IYNAICPCKHCNKCKPGLLAN (IN-21) respectively. Preliminary studies on the mechanisms underlying the insulinotropic actions of peaks 21, 22, 23 and 24 suggest possible involvement of a cAMP-dependent, G protein-insensitive pathway. These data indicate that Bombina variegata skin secretions contain peptides with insulin-releasing activity, which may have mammalian counterparts and prove useful for possible exploitation as antidiabetic agents from natural resources.