新发现的胰脏活性多肽

胰脏中除了人们所熟悉的胰岛素、胰高血糖素、生长激素释放抑制因子和胰多肽外,近几年又陆续发现了几个新的胰脏活性多肽。它们是甘丙肽(galanin),胰脏释放抑制因子(pancreastatin),胰岛淀粉样多肽(islet amyloid polypeptide)及胰岛素拮抗肽。本文着重介绍它们的结构与功能。对这些活性多肽的深入研究,无疑在理论上或临床实践中均将有其重要的意义。     

重组人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白的生物学活性检测

目的：评价原核表达、纯化的6×His-硫氧还蛋白（TRX）一人肿瘤坏死因子α（TNFα）抑制肽-C端抗炎酸性尾巴融合蛋白的生物学功能。方法：在大肠杆菌中分别表达带His标签的TRX对照蛋白及TRX蛋白融合的人TNFα抑制肽-抗炎酸性尾巴融合蛋白,并对2种蛋白进行N^2＋金属螯合层析纯化,采用MTT法检测纯化后的蛋白及化学合成多肽抑制TNFα标准品对L929细胞的细胞毒活性。结果：与对照蛋白相比,融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成多肽均能拮抗TNFα标准品对L929细胞的细胞毒作用。结论：融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成肽均能有效拮抗TNFα的生物学作用,为今后发展抑制TNFα为主的抗炎生物药物奠定了基础。     

利用噬菌体展示技术筛选胰岛素模拟肽

为了寻找能够模拟胰岛素生物活性的小肽,以胰岛素多克隆抗体为靶标,筛选噬菌体展示随机C7C环肽库.3轮筛选后,通过ELISA方法挑取与靶分子特异性结合的15个阳性克隆,测序获得两条序列,分析所得序列并合成相应短肽.通过细胞生物学活性检测,小肽CPTSQANSC（ZJ1）能够竞争性的抑制胰岛素与其受体的结合,并对正常小鼠和四氧嘧啶诱导的糖尿病小鼠,都有明显的降血糖作用.上述结果表明,小肽CPTSQANSC具有胰岛素样生物学活性.而小肽CVQPSHSSC（ZJ2）表现出胰岛素拮抗活性,能引起正常小鼠血糖升高.这表明筛选到了能够模拟胰岛素表位的短肽CPTSQANSC,可能为治疗胰岛素依赖性糖尿病提供了新线索.     

核桃多肽对高糖诱导HepG2胰岛素抵抗模型的影响

摘 要：目的：探究核桃多肽在细胞水平上的降糖作用机制,为核桃资源开发利用提供科学依据。方法：在实验室提取的HT多肽,分离纯化HT-1、HT-2的基础上,通过GLUT4转膜活性筛选,L6肌管细胞葡萄糖摄取活性筛选以及高糖诱导HepG2细胞模型胰岛素抵抗实验,探究核桃多肽的降糖生物活性及降糖作用机制。结果：HT多肽、HT-1、HT-2均有一定的促GLUT4转膜活性,其中量效曲线趋势分布较好的为HT-2,15min开始响应,30min达峰值,在25min细胞膜上GLUT4增加1倍;葡萄糖摄取活性都较好,摄取率分别为1.16、1.06和1.36;IRβ、IRS-1蛋白、GLUT2蛋白的表达水平与HT-2呈浓度依赖性增加,表明HT-2通过胰岛素信号传导途径改善葡萄糖代谢。结论：HT多肽、HT-1、HT-2的降糖活性都较好,其中HT-2效果明显,且是通过胰岛素信号传导途径改善葡萄糖代谢。     

慢性高剂量胰岛素刺激猪脂肪细胞脂肪分解

为研究慢性高剂量胰岛素对猪脂肪细胞脂肪分解的影响及其分子机制, 分化的猪脂肪细胞在PKA(Protein kinase A)或ERK(Extracellular signal-related kinase)抑制剂预处理或不处理的情况下, 再用不同浓度的胰岛素(0、200、400、800、1600 nmol/L)处理不同时间(24、48、72、96 h), 通过测定甘油释放量检测脂肪细胞的脂解率; 采用RT-PCR和Western blotting检测perilipin A和PPARg2的mRNA和蛋白表达。结果显示, 慢性高剂量胰岛素以剂量和时间依赖性的方式刺激猪脂肪细胞的脂肪分解, 并削弱脂肪细胞对异丙肾上腺素刺激的脂解应答; 同时显著下调perilipin A和PPARg2的mRNA及蛋白表达; 另外, PKA和ERK抑制剂均显著抑制胰岛素刺激的脂肪分解, 但仅ERK抑制剂显著逆转perilipin A基因表达的下调。由此推测, 慢性高剂量胰岛素通过ERK通路抑制perilipin A的表达, 进而刺激猪脂肪细胞的脂肪分解。     

脂肪细胞补体相关蛋白30的研究进展

脂肪细胞补体相关蛋白30(Acrp30)是仅由脂肪细胞合成和分泌的一种激素,与机体胰岛素敏感性有密切关系,可降低餐后血游离脂肪酸水平并加强胰岛素抑制肝葡萄糖输出的作用,同时还有抗动脉粥样硬化的特性。     

猪胰岛素前体在酵母Kluyveromyces lactis中的分泌…

通过对包括猪胰岛素前体（ＰＩＰ）基因在内的表达框架克隆至质粒ｐＫＤ１衍生的两种载体上而在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓ ｌａｃｔｉｓ中分泌表达猪胰岛素前体。根据放射免疫测定结果,猪胰岛素前体的表达水平为２０－３０ｍｇ／Ｌ,猪胰素胶体经过胰肽被转变基因工程人胰岛素,分析结果表明,来自Ｋ．ｌａｃｔｉｓ的人胰岛素,其氨基酸组成、晶体形状和生物活力天然胰岛素相同。     

硒化合物的拟胰岛素作用

硒的主要功能是它作为硒酶或硒蛋白的活性成分 ,能够清除自由基 ,保护细胞膜免于氧化 ,增强机体免疫的作用 ;此外 ,硒还能拮抗汞、砷、镉、铊的毒性。近年来 ,研究还发现 :具有降低血糖和调控胰岛素介导的代谢过程等拟胰岛素作用。1 .增强葡萄糖的运输和转化Ｏｓａｍｕ等[1] 报道了在大鼠脂肪细胞中 ,硒与胰岛素一样具有增强葡萄糖的转运能力 ,硒和胰岛素都能够刺激增强从胞内到质膜上的两个葡萄糖转运蛋白 (ｔｒａｎｓｐｏｒｔｅｒ,ＧＬＵＴ)移位活性 ,从而达到加速葡萄糖的运输 ,降低机体血糖的作用。研究还发现硒能够刺激增强每一个与胰…     

肥胖与糖尿病的连结点——胰岛素抗性蛋白

在研究抗糖尿病新药TZDS的作用机制发现胰岛素抗性蛋白（resistin),它是由脂肪细胞分泌的脂肪细胞特异性多肽。在肥胖小鼠中,胰岛素抗性蛋白与高血糖症和胰岛素抗性有关。在胰岛素抵抗和高血糖症中,胰岛素抗性蛋白升高,如果给予胰岛素抗性蛋白抗体则血糖下降,对胰岛素的敏感性也得到恢复。这表明胰岛素抗性蛋白可能与肥胖和糖尿病有关,可能为两者之间的连结点。     

体外抑制鸡传染性支气管炎病毒的多肽鉴定

使用纯化的鸡传染性支气管炎病毒(IBV), 从12肽噬菌体随机展示肽库中筛选特异性结合多肽. 经过四轮淘筛, 得到10个阳性噬菌体, 进一步进行测序、血凝抑制活性及病毒抑制特性鉴定. 所有阳性噬菌体均能特异性阻断IBV对HeLa细胞的感染, 并能抑制IBV对鸡红细胞的血凝活性. 人工合成其中一条病毒抑制效价最高的线性多肽“GSH HRH VHS PFV”, 其细胞抑制试验证实该多肽同样具有病毒抑制特性. 以上结果为研制抗病毒分子及鉴定病毒与细胞相互作用功能域等奠定了基础.     

Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action.

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Ssp dnaB蛋白质内含子介导的重组人脑钠素的制备

脑钠素(BNP)是临床治疗代偿失调性心衰竭的有效药物。将脑钠素与组氨酸标签(His-tag)以及具有自我剪切功能的Ssp dnaB微型蛋白质内含子进行融合表达。表达产物经Ni-Sepharose亲和层析及体外复性处理后,用CM_纤维素对复性产物进行了浓缩,并通过改变CM-纤维素柱内的pH及温度,诱导Ssp dnaB微型蛋白质内含子的剪切作用,使脑钠素从融合蛋白中释放并与载体蛋白(His-DnaB)分离,再经C4反相高效液相色谱法进一步纯化后,从每升培养液中获得了2.8mg纯度达97%的重组人脑钠素。体外活性测定结果表明,重组人脑钠素对兔胸主动脉条具有显著的血管舒张效应,其EC50为1.94×10-6mg/mL。     

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10(-9) M glucagon, and at 3 x 10(-7) M glucagon insulin release was enhanced 290% above the glucose base line.     

Insulin releasing activity of gastrointestinal glucagon-like immunoreactive materials in perfused rat pancreas

Insulin-releasing activity of porcine gastrointestinal glucagon-like immunoreactive materials purified by affinity chromatography was examined in the perfused rat pancreas. When glucose concentration of the perfusate was raised from 60 to 100 mg/dl, augmented insulin release was observed. The mean incremental area of immunoreactive insulin (sigma delta IRI) during the first 10 min thus observed was 19.07 +/- 3.76 ng/10 min. Pancreatic glucagon and the extract from the gastric fundus showed the enhancement of insulin release in this system when they were added to the perfusate at the rate of 100 ng/min for 5 min; delta IRI were 41.92 +/- 8.47 and 71.70 +/- 18.09 ng/10 min, respectively, which were significantly higher than that of 100 mg/dl of glucose alone. However, no significant difference in the insulinogenic activity was noticed between the extracts from the small intestine and the control. These results suggest that the extract from the gastric fundus has insulinogenic activity similar to that of pancreatic glucagon.     

Purification and characterization of an immunomodulatory Peptide from bovine placenta water-soluble extract

An immunomodulatory peptide was isolated from bovine placenta water-soluble extract and purified by consecutive chromatography on DEAE Sepharose CL-6B, Sephadex G-25, and Sephasil C18 column using lymphocyte proliferation assay to identify the active fractions. The immunomodulatory peptide showed a dose-dependent stimulating effect on lymphocyte proliferation. The isoelectric point of the immunodulatory peptide was determined to be 3.82 by capillary isoelectric focusing electrophoresis. The molecular mass of the immunomodulatory peptide was determined to be 2133.52 Da by mass spectrometry. The first 10 amino acid sequence of the immunomodulatory peptide was Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr. This immunomodulatory peptide showed no significant homology with other immunomodulatory peptides. Additionally, it was thermostable, retaining about 60% of immune activity after incubating at 80 degrees C for 30 min.     

Purification of a bacteriolytic enzyme from Bacillus sp. active against Streptococcus mutans

Bacillus sp. strain YU-1006, which was isolated from soil, produced a bacteriolytic enzyme which was active against Streptococcus mutans KCTC 3283. The enzyme was purified 60 fold with a 6% yield by CM-cellulose column chromatography and CM-Sephadex G-50 column chromatography. Lytic activity was stable in the range of pH 6.0~9.0 up to 42°e active enzyme is a 24,000 dalton monomer as estimated by SDS-PAGE.     

Dihydrokaempferol Glucoside from Cotyledons Promotes Flowering in Pharbitis nil

An extract of cotyledons of Pharbitis nil, which had been exposedto short-day conditions, was tested for flower-promoting activityin a shoot-tip assay system in vitro. The crude extract hadno flower-promoting activity, however, after partitioning ofthe crude extract with dichloromethane, the resulting aqueousfraction had flower-promoting activity. This activity was separatedinto two fractions by column chromatography on Toyopearl HW-40.One active fraction was identified as dihydrokaempferol-7-O-rß-D-glucoside(DHK-glc). This compound exhibited flower-promoting activityat the extremely low concentration of 4.4x10^-9. (Received April 25, 1995; Accepted August 11, 1995)     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

哺乳类(兔)红细胞分化因子的纯化及特性分析

本文报道从哺乳动物(兔)红细胞分离、提纯红细胞分化因子(ErythroidDifferentiation Factor,简称EDF及对其性质的分析。按Bishop方法给新西兰兔注射盐酸苯肼(2.5％)0.3mL/kg体重/日连续6days致贫血,收集血液用生理盐水洗去白细胞后,得到的网织红细胞用冻融法使细胞裂解,用乙醇、氯仿选择性变性除去血红蛋白,凝胶过滤,连续经DEAE-纤维素及CM-纤维素柱层析,取得较原裂解液纯化了31 000倍的活性物质EDF,产率为9.1％,分子量15kD,在SDS-PAGE电泳图中为单一蛋白带。EDF对热相对稳定,100℃处理10min仍保持一定活性,对蛋白酶敏咸而对DNA酶I及RNA酶不敏感。EDF对体外培养细胞的生长抑制作用已在体外实验体系得到验证。