信号肽疏水性的提高促进青霉素G酰化酶分泌

设计和合成了一段具有连续１０仆亮氨酸强疏水核心的信号肽（ａｒｔｉｆｉｃｉａｌ ｓｉｇｎａｌ ｐｅｐｔｉｄｅ,ＡＳＰ）,由ＥｃｏＲＩ－ＫｐｎＩ位点融合到青霉素Ｇ酰化酶（ｐｅｎｉｃｉｌｌｉｎ Ｇａｃｙｌａｓｅ,ＰＡＣ）信号肽（ｗｉｌｄ ｔｙｐｅｓｉｇｎａｌ ｐｅｐｔｉｄｅ,ＷＴＳＰ）的－４Ｐｒｏ位点,分别构建了ＰＡＣ表达质粒：ｐＫＫｐａｃ△ＳＰ,ｐＫＫｐａｃＷＴＳＰ,ｐＫＫｐａｃＡＳＰ,ｐＥＴｐａｃ     

利钠肽的结构、受体与生理作用

利钠肽 (ｎａｔｒｉｕｒｅｔｉｃｐｅｐｔｉｄｅ ,ＮＰ)是近 2 0年才发现的一类多肽 .到目前为止 ,人类共发现了 5种利钠肽 ,即心房利钠肽 (ａｔｒｉａｌｎａｔｒｉｕｒｅｔｉｃｐｅｐｔｉｄｅ ,ＡＮＰ )、脑利钠肽(ｂｒａｉｎｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅ,ＢＮＰ)、Ｃ型利钠肽 (Ｃ ｔｙｐｅｎａｔｒｉｕｒｅｔｉｃｐｅｐｔｉｄｅ ,ＣＮＰ)、Ｖ型利钠肽 (ｖｅｎｔｒｉｃｌｅｎａｔｒｉｕｒｅｔｉｃｐｅｐｔｉｄｅ,ＶＮＰ)和Ｄ型利钠肽 (ｄｅｎｄｒｏａｓｐｉｓｎａｔｒｉｕｒｅｔｉｃｐｅｐｔｉｄｅ ,ＤＮＰ) .ＶＮＰ和ＤＮＰ至今…     

蝎毒抗癫痫反复发作的GFAP基因调控机制

已有的资料证实红藻氨酸 (Ｋａｉｎｉｃａｃｉｄ ,ＫＡ)癫痫模型具有癫痫发作敏感性长期增强的特征 ,伴有海马结构胶质原纤维酸性蛋白 (ｇｌｉａｌｆｉｂｒｉｌｌａｒｙａｃｉｄｐｒｏｔｅｉｎ ,ＧＦＡＰ)的大量表达 ,其为海马结构反应性胶质化的神经病理学基础 ;用反义ＧＦＡＰｍＲＮＡ转染的星形胶质细胞的细胞株内不再表达ＧＦＡＰ。新近 ,我们从ＫＡ癫痫大鼠海马结构中发现调控ＧＦＡＰ基因表达的序列特异的ＤＮＡ结合蛋白。我们从前的工作已证实蝎毒具有明显的抗癫痫反复发作的作用 ,抑制癫痫大鼠海马结构ＧＦＡＰ的过量表达 ,但蝎毒阻…     

免疫亲和层析法纯化单链尿激酶型纤溶酶原激活剂

尿激酶原 (Ｐｒｏ ｕｒｏｋｉｎａｓｅ ,ｐｒｏ ＵＫ) ,也称单链尿激酶型纤溶酶原激活剂 (Ｓｉｎｇｌｅ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｓｃｕ ＰＡ) ,与ｔ ＰＡ一样是第二代溶栓药物。给药时 ,保持无活性的酶原状态 ,只激活被纤维蛋白吸附的纤溶酶原 ,而对游离的纤溶酶原没有作用 ,即只在血栓表面才能活化转变为双链尿激酶 (Ｔｗｏ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｔｃｕ ＰＡ或ＵＫ) ,因而具有较高的特异性溶血栓作用[1 ] 。尿激酶原…     

肺血管EDNO及其合酶在大鼠低氧性肺动脉高压形成中的作用

本研究观察了低氧对大鼠肺组织和血管内皮一氧化氮合酶（ＮＯＳ）活性及内皮衍生一氧化氮（ＥＤＮＯ）依赖性舒张反应的影响,以及ＮＯＳ抑制剂（Ｌ－ＮＡＭＥ）对常氧和低氧大鼠肺组织和血管内皮ＮＯＳ活性及颈、肺动脉血压（ＣＡＰｓ、ｍＰＡＰ）的作用。结果表明常氧大鼠肺泡内无肌性血管内皮未见ＮＯＳ活性,其肺血管床对ＥＤＮＯ依赖性舒血管物质ＢＫ没有反应,注射Ｌ－ＮＡＭＥ后大鼠ｍＰＡＰ略有降低,ＣＡＰｓ有所升高。低氧大鼠肺泡内无肌性血管内皮显示ＮＯＳ活性,对ＢＫ的ＥＤＮＯ依赖性舒张反应呈剂量依赖性增大,注射Ｌ－ＮＡＭＥ使低氧大鼠ｍＰＡＰ显著降低（Ｐ＜０．０１）,ＣＡＰｓ显著升高（Ｐ＜０．０５）。提示肺血管ＥＤＮＯ及其合酶在维持正常成年大鼠肺循环低压低阻中的生理作用值得进一步探讨;低氧引起肺血管内皮ｅｃＮＯＳ活性增加和ＥＤＮＯ生成增多可能起到限制肺动脉压过度升高的调制作用,也可能对肺血管内皮产生毒性作用,反而促进肺动脉高压的发生和发展。     

全蝎抗癫痫发作敏感性的阿片肽机制

红藻氨酸（ＫａｉｎｉｃＡｃｉｄ,ＫＡ）癫痫模型,探讨全蝎抗癫痫发作敏感性长期增强的阿片肽机制。本实验选用ＳＤ大鼠,随机分为两组,分别给予生理盐水（ＮＳ）和全蝎粗提液灌胃１０天,１０天后两组均分别颈部皮下注射ＮＳ和惊厥剂量（１０ｍｇ／ｋｇ）的ＫＡ,再分别继续给予ＮＳ和全蝎粗提液灌胃１０天后,用阈下剂量（５ｍｇ／ｋｇ）的ＫＡ检测癫痫敏感性;用Ｆｏｓ免疫反应活性检测海马结构中神经元的兴奋性;用原位杂交技术检测海马脑啡肽原（ＰＥＮＫ）ｍＲＮＡ的动态变化过程。结果显示：实验对照组大鼠癫痫行为敏感性明显增强,脑内癫痫敏感性相关脑区海马齿状回颗粒细胞（ＤＧＣｓ）ｃ－Ｆｏｓ免疫反应阳性细胞数目明显增加,同时海马内具有致癫痫作用的脑啡肽原（ＰＥＮＫ）ｍＲＮＡ表达也明显增加;而实验组动物未见上述改变。本工作证实中药全蝎有明显降低海马神经元兴奋性及抗癫痫发作敏感性形成的作用,并提示这很可能与其抑制ＰＥＮＫｍＲＮＡ表达增加有关     

大鼠运动性肥大心脏心肌血管内皮生长因子及其基因表达的研究

目的和方法：血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）是新近确定的一种特异作用于血管内皮细胞的活性肽。最近发现正常心肌细胞可表达ＶＥＧＦ,高血压肥大心脏心肌ＶＥＧＦ及基因表达增强,但对运动性心肌肥大时的变化尚不清楚。本实验采用免疫组化和分子杂交方法,对游泳运动１０周大鼠稳定期肥大心脏心肌ＶＥＧＦ及其基因表达进行研究。结果：ＷｉｓｔａｒＫｙｏｔｏ（ＷＫＹ）大鼠、自发性高血压大鼠（ｓｐｏｎｔａｎｅｏｕｓｌｙｈｙｐｅｒｔｅｎｓｉｖｅｒａｔｓ,ＳＨＲ）和运动大鼠心肌细胞浆内均有特异性ＶＥＧＦ染色颗粒,但运动大鼠心肌细胞胞浆内染色颗粒增加最明显。Ｎｏｒｔｈｅｒｎ分子杂交结果表明三组大鼠心肌均有ＶＥＧＦｍＲＮＡ表达,其中ＳＨＲ表达最强,运动大鼠比ＷＫＹ大鼠增强,但低于ＳＨＲ。结论：目前对这一结果的生理意义还不清楚,推测可能与心肌肥大时细胞间质血管增生有关。     

阿尔茨海默病相关的早老蛋白与Notch和Wnt信号通路

自 1 995年发现高度同源的早老蛋白(ｐｒｅｓｅｎｉｌｉｎ ,ＰＳ) 1、2基因突变可导致家族性早发型阿尔茨海默病 (Ａｌｚｈｅｉｍｅｒｄｉｓｅａｓｅ ,ＡＤ)以来 ,目前不仅已证实ＰＳ参与老年斑核心物质β 淀粉样蛋白 (β ａｍｙｌｏｉｄ ,βＡ)的形成 ,还发现ＰＳ是一种多功能蛋白质 ,与细胞内的多种信号转导通路有关。ＰＳ介导Ｎｏｔｃｈ、Ｗｎｔ/Ｗｉｎｇｌｅｓｓ信号转导的机制、及其与ＡＤ病理改变的关系已成为ＡＤ研究领域的一个新的关注点。1 .ＰＳ和Ｎｏｔｃｈ信号通路的关系Ｎｏｔｃｈ信号在神经元发育中有重要作用 ,通过旁侧抑制…     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)~+RNA from cat dorsal root ganglion

ＴａｃｈｙｋｉｎｉｎｆａｍｉｌｙｉｓａｇｒｏｕｐｏｆｎｅｕｒｏｐｅｐｔｉｄｅｓｗｉｔｈｓｉｍｉｌａｒＣｔｅｒｍｉｎａｌｓｅｑｕｅｎｃｅｓａｎｄｒｅｌａｔｅｄｂｉｏａｃｔｉｖｉｔｉｅｓ.ＴｈｅｍａｊｏｒｔａｃｈｙｋｉｎｉｎｓｉｎｍａｍｍａｌｉａｎａｒｅｓｕｂｓｔａｎｃｅＰ(ＳＰ),ｎｅｕｒｏｋｉｎｉｎＡ(ＮＫＡ)ａｎｄｎｅｕｒｏｋｉｎｉｎＢ(ＮＫＢ).Ｃｏｒｒｅｓｐｏｎｄｉｎｇｔｏｔｈｅｓｅｐｅｐｔｉｄｅｓ,ｔｈｒｅｅｄｉｓｔｉｎｃｔｔａｃｈｙｋｉｎｉｎｒｅｃｅｐｔｏｒｓｗｅｒｅｄｉｓｃｏｖｅｒｅｄａｎｄｎ…     

微生态调节剂抗癫痫敏感性形成作用中神经胶质原纤维酸性蛋白免疫活性变化

用红藻氨酸（ＫａｉｎｉｃＡｃｉｄ,ＫＡ）１２ｍｇ／ｋｇ给ＳＤ大鼠颈部皮下注射,诱发动物出现癫痫发作,该癫痫发作于８小时内完全缓解。ＫＡ后１周再次给予ＫＡ（此次为阈下剂量５ｍｇ／ｋｇ）,检测上述动物对癫痫刺激的敏感性。结果表明,与对照组比较,于４周前开始并连续灌服微生态调节剂实验组动物癫痫敏感性的形成受到明显抑制（Ｐ＜０００１）,同时用免疫细胞化学方法观察大鼠脑内海马部位星形胶质细胞的神经胶质原纤维酸性蛋白（ＧｌｉａｌＦｉｂｒｉｌａｒｙａｃｉｄｉｃｐｒｏｔｅｉｎＧＦＡＰ）免疫反应活性的变化,发现ＫＡ后１～７天,实验组与对照组比较,海马部位神经胶质原纤维酸性蛋白免疫反应活性明显受到抑制。结果表明微生态调节剂的抗癫痫敏感性作用可能与抑制胶质细胞过度增生有关。     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

Stereoselective interactions of peptide inhibitors with the beta-amyloid peptide

Residues 16-20 of the beta-amyloid peptide (A beta) function as a self-recognition element during A beta assembly into fibers. Peptides containing this motif retain the ability to interact with A beta and, in some cases, potently inhibit its assembly. Replacing L- with D-amino acids could stabilize such peptides and permit their evaluation as therapeutic agents for Alzheimer's disease. Here we have assessed the effect that such a chiral reversal has on inhibitory potency. D-enantiomers of five peptides, KLVFFA, KKLVFFA, KFVFFA, KIVFFA, and KVVFFA, were unexpectedly more active as inhibitors in an in vitro fibrillogenesis assay. Circular dichroism showed that D-KLVFFA more effectively prevented A beta adopting the beta-sheet secondary structure correlated with fibrillogenesis. Electron microscopy showed that fiber formation was also more strongly inhibited by D-KLVFFA. Heterochiral inhibition was confirmed using D-A beta, on the principle that enantiomeric proteins exhibit reciprocal chiral biochemical interactions. With D-Abeta, L-KLVFFA was the more potent inhibitor, rather than d-KLVFFA. Most significantly, D-peptides were more potent at reducing the toxicity of both A beta1-40 and A beta 1-42 toward neuronal cells in culture. This unforeseen heterochiral stereoselectivity of A beta for D-peptide inhibitors should be considered during future design of peptide-based inhibitors of A beta neurotoxicity and fibrillogenesis.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction

Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG). Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG. Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase. While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA. PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported. Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation. Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor. PKG translocation is ANP-dependent but not nitric oxide-dependent. Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects. As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.