A8—10替换的胰岛素似物的合成及生物活性

本文报道了用Ｆｍｏｅ固相法合成３种胰岛素Ａ链小环（Ａ８－１０）被不同碱性氨基酸取代的Ａ链类似物,并分别与天然胰岛素Ｂ链重组成相应胰岛素类似物;经受体结合,整体活性及抗体结合实验,均表现出相应的活性。从中可以推测出：Ａ链小环区域不是胰岛素表现生物活性的重要部位,而是胰岛素与其抗体结合较重要的区域。     

神经肽AVP（4—8）激活大鼠海马突触膜上的G蛋白偶联受体

精氨酸加压素（ＡＶＰ）的Ｃ端内片段,ＡＶＰ（４－８）,具有增强记忆的功能,它在大鼠脑内引发一系列的生理和生化变化。ＧＴＰ－结合调节蛋白（Ｇ蛋白）介导多数神经肽和神经调质的细胞内生理生化反应,放射性受体结合实验显示,在海马突触膜上存在ＡＶＰ（４－８）的特异性结合位点。ＡＶＰ（４－８）在海马突触膜上的结合能够进一步刺激ＧＴＰγＳ结合并可被ＡＶＰ（４－８）的受体拮抗剂ＺＤＣ（Ｃ）ＰＲ所逆转。从以上实验结     

胰岛素样生长因子结合蛋白—1（IGFBP—1）研究进展

胰岛素样生长因子结合蛋白-1（IGFBP-1）参与生长、发育、生殖及血糖等生理过程,它是一个多功能蛋白,主要通过与IGF-1结合发挥作用,在IGFBP-1蛋白质结构中,N端是与IGF-1结合的重要区域,中心区决定了IGFBP的特异性,C端部分参与了与IGF-1结合并与细胞粘附相关。IGFBP-1是否磷酸化对其生物学作用有着重要影响。IGFBP-1基因转录受到多种转录因子共同调控,其中胰岛素是其主要的调控因子。     

从基因工程小C肽人胰岛素原类似物（B—R2—A）制备人胰岛素

以融合蛋白的形式,在Ｅｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原来似物（Ｂ－Ｒ２－Ａ）,表达的融合蛋白可占细胞总蛋白６８％。经磺酸解,及初步分离Ｓ－磺酸型融合蛋白,再经ＣＮＢｒ裂解后,进行还原重组,ＨＰＬＣ分离纯化等步骤,每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－Ｓｅｐｈａｄｅｘ－Ａ２５纯化,得重组人胰岛素约２０ｍｇ,基氨基酸组成与人胰岛素相同,并具有与猪胰岛素相同的生物活性。     

[B3—Lys]—胰岛素的研究：受体结合及生物活性

本文用＾１２５Ｉ－［Ｂ３－Ｌｙｓ］－胰岛素和＾１２５Ｉ－胰岛素研究了［Ｂ３－Ｌｙｓ］－胰岛素和胰岛素与人胎盘细胞膜胰岛素受体结合物性并进行了比较。实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素受体结合能力比天然猪胰岛素高。由竞争取代曲线得到的［Ｂ３－Ｌｙｓ］－胰岛素和猪胰岛素的ＩＣ（５０）值分另为０．６５和１．１１ｎｍｏｌ／Ｌ。经Ｓｃａｔｃｈａｒｄ分别得出［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素     

胰岛素B链N端的修饰及其生物活性变化

通过对在大肠杆菌体系中表达及部分加工的粗产物的反相快速蛋白液相色谱的分离,我们得到了Ｌ－Ｍｅｔ~（ＢＯ）－人胰岛素原、Ｌ－Ｍｅｔ~（Ｂ－１）－Ｌ－Ｌｙｓ~（ＢＯ）－人胰岛素原,氨基酸组成分析结果与设想的完全一致。两者的胰岛素受体活性分别为人胰岛素原的６６％及５４％,而胰岛素的放射免疫活性分别降为人胰岛素原的２２％及９％。表明胰岛素Ｂ链的Ｎ端对胰岛囊分子与其受体的结合有一定的重要性,而更重要的是它非常可能参与组成胰岛素分子的抗原决定簇。     

AVP（4—8）增强CK—N—XH细胞内PKC和MAPK的活性

ＡＶＰ（４￣８）是精氨酸加压素（ＡＶＰ）在脑内的天然酶解产物,具有增强学习记忆的功能。为了进一步阐明其作用的分子机制,以ＳＫ－Ｎ－ＳＨ成神经瘤细胞（ＳＫ细胞）为模型进行研究。放射性配基结合实验表明,在ＳＫ细胞上存在ＡＶＰ（４￣８）的特异性结合位点。ＡＶＰ（４￣８）可以刺激ＳＫ细胞中蛋白激酶Ｃ（ＰＫＣ）和促细胞分裂原活化的蛋白激酶（ＭＡＰＫ）尖性的升高,并可以被ＡＶＰ（４￣８）的受体拮抗剂ＺＤＣ（Ｃ     

AVP_（4－8）结合点在大鼠海马内的分布和AVP_（4－8）对其受体发育的影响：放射自显影研究

本文利用放射自显影方法结合神经毒对海马神经元的选择性损毁观察ＡＶＰ（４－８）结合点在大鼠海马内的分布和定位;利用外源性ＡＶＰ（４－８）对新生大鼠的处理,观察海马ＡＶＰ（４－８）结合点的发育调节。在成年大鼠海马内,ＡＶＰ（４－８）结合点集中分布在整个海马的锥体细胞层和齿回的颗粒细胞层。秋水仙碱处理后,齿回颗粒细胞层消失,齿回区的ＡＶＰ（４－８）结合点也消失。红藻氨酸（Ｋａｉｎｉｃａｃｉｄ）处理后海马ＣＡ３－ＣＡ４的锥体细胞层消失,该区的ＡＶＰ（４－８）结合点也消失。新生大鼠海马锥体细胞层的ＡＶＰ（４－８）结合点在出生后第６天开始出现,齿回颗粒细胞层的ＡＶＰ（４－８）结合点在出生后第７天开始出现。然而,新生大鼠每天经外源性ＡＶＰ（４－８）处理,海马锥体细胞层和齿回颗粒细胞层的结合点均在出生后第５天已变得十分稠密。本文就大鼠海马ＡＶＰ（４－８）结合点的特异性分布和ＡＶＰ（４－８）处理促进海马ＡＶＰ（４－８）结合点的发育与成年后大鼠学习能力的提高的相互关系作了讨论。     

[A_(15)Asn,A_(17)Pro,A_(21)Ala]-胰岛素的体内外生物活性

对胰岛素的研究使得许多生命科学中的重要问题被认识．然而对于胰岛素结构和功能关系,仍有许多不清楚的地方．在胰岛素Ａ链的Ｎ端和Ｃ端各有一个螺旋区：Ａ２－８和Ａ１２－１８．杨士珍［１］的研究表明：缺失Ａ１２－１８肽段的Ａ链类似物虽然能和天然Ｂ链重组,但其重组产物只保留了天然胰岛素的约６％的体内外生物活性．这说明Ａ１２－１８对胰岛素的生物活性十分重要．但是这一结果还不能说明是Ａ１２－１８螺旋还是Ａ１２－１８肽段对胰岛素的生物活性十分重要．根据Ｃｈｕｏ－Ｆａｓｍａｎ法,在含有Ａｓｎ和Ｐｒｏ的肽段中不能形成…     

Design and Synthesis of Potent Tyr(OMe)5-Gonadotropin-Releasing Hormone (GnRH) Analogues with Modifications at Positions 6, 9 and 10

Summary We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilicd-amino acids such asd-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

3个六肽的胰岛素受体结合和体内生物活性

为了研究胰岛素受体结合部位的结构和功能,设计并用固相方法合成了３个六肽．在浓度大于１×１０３ｎｍｏｌ／Ｌ时,ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ）具有明显的胰岛素受体结合活力;Ｈ－Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ－ＯＨ的这一活力则不明显;而Ｈ－Ｇｌｙ－Ｇｌｕ－Ａｒｇ－Ｇｌｙ－Ｐｈｅ－Ｐｈｅ－ＯＨ则增强胰岛素和其受体的亲和性．然而,它们都没有体内生物活性．这表明：环六肽部分模拟了胰岛素受体结合部位的空间构象;胰岛素受体结合部位的疏水性和其中的Ｂ２３Ｇｌｙ－Ｂ２４Ｐｈｅ－Ｂ２５Ｐｈｅ对胰岛素和其受体的结合起重要作用．     

Tiered Assembly of the Yeast Far3-7-8-9-10-11 Complex at the Endoplasmic Reticulum

Target of rapamycin signaling is a conserved, essential pathway integrating nutritional cues with cell growth and proliferation. The target of rapamycin kinase exists in two distinct complexes, TORC1 and TORC2. It has been reported that protein phosphatase 2A (PP2A) and the Far3-7-8-9-10-11 complex (Far complex) negatively regulate TORC2 signaling in yeast. The Far complex, originally identified as factors required for pheromone-induced cell cycle arrest, and PP2A form the yeast counterpart of the STRIPAK complex, which was first isolated in mammals. The cellular localization of the Far complex has yet to be fully characterized. Here, we show that the Far complex localizes to the endoplasmic reticulum (ER) by analyzing functional GFP-tagged Far proteins in vivo. We found that Far9 and Far10, two homologous proteins each with a tail-anchor domain, localize to the ER in mutant cells lacking the other Far complex components. Far3, Far7, and Far8 form a subcomplex, which is recruited to the ER by Far9/10. The Far3-7-8- complex in turn recruits Far11 to the ER. Finally, we show that the tail-anchor domain of Far9 is required for its optimal function in TORC2 signaling. Our study reveals tiered assembly of the yeast Far complex at the ER and a function for Far complex''s ER localization in TORC2 signaling.     

Review ArticleNaphthalene Combretastatin Analogues: Synthesis,Cytotoxicity and Antitubulin Activity

Synthesis and evaluation of new combretastatin analogues with varied modifications on the bridge and the aromatic rings, have shown that the 2-naphthyl moiety is a good surrogate for the 3-hydroxy-4-methoxyphenyl (B-ring) of combretastatin A-4. Other bicyclic systems, such as 6(7)-quinolyl and 5-indolyl, can replace the B-ring, but they produce less potent analogues in the cytotoxicity and tubulin polymerization inhibition assays. Other modifications are detrimentral for the potency of the studied analogues. The 2-naphthyl combretastatin 53 and the related 6-quinolyl combretastatin 106 analogues are the most potent among the derivatives of this type, whereas 92 and 95 are the most potent among the naphthalene derivatives with a heterocycle in the bridge. Previous and new results in this family of combretastatin analogues are discussed.     

Synthesis of Methylenebis(Phosphonate) Analogues of 2-, 4-, and 6-Pyridones of Nicotinamide Adenine Dinucleotide

The synthesis of metabolically stable methylenebis(phosphonate) analogues of 2-, 4-, and 6-pyridones of nicotinamide adenine dinucleotide (NAD) is reported. In contrast to natural pyrophosphates, these NAD analogues are able to penetrate the cell membrane and can be used as probes in cellular assays.     

Quantification of isomeric equilibria formed by metal ion complexes of 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine (8,8aPMEA) and 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine (9,8aPMEA). Derivatives of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)

The acidity constants of the two-fold protonated acyclic 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(9,8aPMEA)(+)(-), and its 8-isomer, 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(8,8aPMEA)(+)(-), both abbreviated as H2(PA)(+)(-), as well as the stability constants of their M(H;PA)+ and M(PA) complexes with the metal ions M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+, have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO3) and 25 degrees C. Application of previously determined straight-line plots of log K(M)M(R-PO3) versus pK(H)H(R-PO3)for simple phosph(on)ate ligands, R-PO3(2-), where R represents a residue without an affinity for metal ions, proves that for all M(PA) complexes a larger stability is observed than is expected for a sole phosphonate coordination of the metal ion. This increased stability is attributed to the formation of five-membered chelates involving the ether oxygen present in the aliphatic residue (-CH2-O-CH2-PO3(2-)) of the ligands. The formation degrees of these chelates were calculated; they vary between about 13% for Ca(8,8aPMEA) and 71% for Cu(8,8aPMEA). The adenine residue has no influence on complex stability except in the Cu(9,8aPMEA) and Zn(9,8aPMEA) systems, where an additional stability increase attributable to the adenine residue is observed and equilibria between four different isomers exist. This means (1) an open isomer with a sole phosphonate coordination, M(PA)op, where PA(2-)=9,8aPMEA2-, (2) an isomer with a five-membered chelate involving the ether oxygen, M(PA)cl/O, (3) an isomer which contains five- and seven-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)cl/O/N3, and finally (4) a macrochelated isomer involving N7, M(PA)cl/N7. For Cu(9,8aPMEA) the formation degrees are 15, 30, 48 and 7% for Cu(PA)op, Cu(PA)cl/O, Cu(PA)cl/O/N3 and Cu(PA)cl/N7, respectively; this proves that the macrochelate involving N7 is a minority species. The situation for the Cu(PMEA) system, where PMEA2- represents the parent compound, i.e. the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine, is quite similar. The relationship between the antiviral activity of acyclic nucleoside phosphonates and the structures of the various complexes is discussed and an explanation is offered why 9,8aPMEA is biologically active but 8,8aPMEA is not.     

改性胶原蛋白膜用于大肠杆菌EP8-10组合固定化

探讨了不同方法对胶原蛋白膜进行改性处理,结果表明经过甲醛处理过的胶原蛋白膜具有良好的通透性及耐水性能。将胶原蛋白膜与卡拉胶凝胶颗粒形成组合固定化体系,用于大肠杆菌EP8-10固定化,天冬氨酸转氨酶活回收率达到了91.3%,反应20批次后,酶活回收率仍在80%以上。     

Synthesis and Biological Activity of 2-Aminopurine Methylenecyclopropane Analogues of Nucleosides

Abstract

Synthesis and biological activity of 7- and 9-isomers (Z+E) of methylenecyclopropane analogues of 2-aminopurine nucleosides is described. The (S,Z)-9-isomer is a substrate for xanthine oxidase.