Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

New indolicidin analogues with potent antibacterial activity*

Abstract: Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH₂, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH₂ in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe^4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.     

参龙降压灵对自发性高血压大鼠血压及血浆ET、CGRP含量的影响

目的与方法本实验观察了中药制剂参龙降压灵对自发性高血压大鼠（ＳＨＲ）血压、血浆内皮素（ＥＴ）及降钙素基因相关肽（ＣＧＲＰ）含量及心室结构的影响,并与尼群地平（ｎｉｆｅｄｉｐｉｎ）及牛黄降压丸作对比。结果各给药组给药１－８周后血压均比ＳＨＲ对照组明显降低（Ｐ＜０.０５－０.０１）,给药６周后参龙降压灵大剂量组血压下降幅度最大,效果优于小剂量组及其他两给药组（Ｐ＜０．０５－０．０１）。各给药组心室结构各参数多有降低,虽无统计学意义（可能例数较少）,但已显示出心室肥厚逆转的趋势。各给药组ＳＨＲ血浆ＥＴ含量均有明显降低,ＣＧＲＰ含量升高（Ｐ＜０.０５－０.０１）,而大剂量参龙降压灵作用更显著,与Ｗｉｓｔａｒ正常组大鼠含量接近。结论参龙降压灵降低ＳＨＲ血压作用确实,有逆转心室肥厚趋势及调节血浆ＥＴ和ＣＧＲＰ含量的作用,且有一定的量效关系。     

含编码人精子膜多肽cDNA和乙肝表面抗原基因的重组痘苗病毒

合成编码一种人精子膜蛋白YWK-Ⅱ胞外区的一段多肽片段的双链寡核苷酸链,HSD-2a.将HSD-2a和3’端乙肝表面抗原主S基因连接并与pUC18质粒重组.经鉴定的重组质粒用BamH Ⅰ和EcoRl酶切后,可分离纯化得到HSD-2a和HBsAg的主S基因连接片段,将该连接片段插入痘苗病毒通用表达载体pGJP-5的痘苗病毒启动子P_(7.5)下游,构建成重组质粒pGJP-HSD/HBs.将该重组质粒传染已感染了天坛痘苗病毒的猴肾细胞CV-1,pGJP-5中含有的TK基因与出发株病毒基因组的TK基因发生体内同源重组,构成重组痘苗病毒vv-HSD/HBs.HuTK~-细胞经vv-HSD/HBs感染后,在5-溴脱氧尿苷存在下进行空斑分析,筛选出TK~-重组病毒.应用ELISA方法对已感染了重组病毒的HuTK~-细胞培养上清和细胞裂解液测定,表明有HSD-2a编码的多肽片段的表达.Western-blot法分析呈现28和30Kd两条显色带.重组痘苗病毒vv-HSD/HBs有可能作为抗生育疫苗用途.     

Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.     

Male mice lacking the gastrin-releasing peptide receptor (GRP-R) display elevated preference for conspecific odors and increased social investigatory behaviors

Previously, we generated gastrin-releasing peptide receptor null mutant mice (GRP-R-deficient mice), and found that these animals displayed increased non-aggressive social responses in an ordinary social interaction test using a resident-intruder method. In the present study, we examined in more detail the social behaviors of GRP-R-deficient male mice. In social interaction tests, GRP-R-deficient mice showed more social responses, such as sniffing and nosing, relative to wild-type mice, and similar results were obtained whether GRP-R-deficient mice served as intruders or residents. In the same way, they showed more contact behaviors toward an anesthetized conspecific, and less locomotor activity than wild-type mice in a social investigation test toward an anesthetized male mouse. Since olfactory systems play important roles in the social behavior of rodents, olfactory preference tests were conducted in order to evaluate the olfactory properties of GRP-R-deficient mice. The results suggest that no differences exist between wild-type mice and GRP-R-deficient mice in the preference between a novel sawdust odor and their own odor, or that of other male mice. However, GRP-R-deficient mice preferred the odor of other male mice to their own, in contrast to wild-type mice. Furthermore, the preferences of GRP-R-deficient and wild-type mice were not disrupted by intraperitoneal infusion of diazepam (1.5 mg/kg). These results indicate that neither the motion, nor the behavior of conspecifics, nor reduced anxiety lead to the increased non-aggressive social responses and/or social investigatory behaviors in GRP-R-deficient mice. Rather, these latter behaviors may be a consequence of altered cognition of conspecific odors in the mutant mice.     

海洋岩虫抗菌肽筛选及抗癌活性的初步研究

将岩虫匀浆液经反相浓缩、凝胶过滤和亲和柱层析获得活性组分，在体外用MTS／PMS(氮兰四唑盐／吩嗪硫酸甲酯)方法筛选抗菌肽，再分别用MTS／PMS方法和ELISA—AFP(甲胎蛋白)方法检测其对人肝癌细胞株HepG2增殖、AFP分泌及对正常大鼠成骨细胞株MC3T3-E1增殖的影响。结果表明，纯化的岩虫抗菌肽为组成型碱性蛋白(MW8．1kDa，pI8．6)，不同浓度抗菌肽对肝癌细胞增殖和AFP的分泌均有抑制作用，抑制作用大小与抗菌肽浓度呈正相关性；对正常成骨细胞未见明显的抑制和杀伤作用。     

一氧化碳对大鼠心室组织心钠素分泌的影响

目的探讨一氧化碳(CO)在大鼠心室组织孵育液中对心钠素(ANP)分泌的影响。方法将正常Wistar大鼠的左、右心室分别分为对照组(未加药)和血红素组(孵育时加入血红素,终浓度10-4mol/L),每组各12例,用Krebs液作为孵育液,放入37℃恒温水浴箱中振荡孵育4 h,用放射免疫法测定孵育液中ANP含量。结果与对照组比较,血红素组大鼠左心室[(9.72±3.59)ng/(g.wet tissue)vs(52.05±31.65)ng/(g.wet tissue),P<0.01]和右心室[(5.55±3.98)ng/(g.wet tissue)vs(62.73±41.66)ng/(g.wet tissue),P<0.01]组织孵育液中ANP含量明显降低。对照组大鼠左右心室组织孵育液ANP含量没有明显差异;血红素组中,大鼠右心室组织孵育液中ANP含量明显低于左心室组织孵育液中ANP含量[(5.55±3.98)ng/(g.wet tissue)vs(9.72±3.95)ng/(g.wet tissue),P<0.05]。结论CO可抑制大鼠左右心室组织孵育液ANP含量,且CO可能对右心室组织孵育液ANP含量的抑制作用大于对左心室组织孵育液ANP含量的抑制作用。     

心力衰竭患儿血浆心房利钠肽水平变化及临床意义

目的:探讨心力衰竭患儿血浆心房利钠肽(ANP)的变化及其临床意义。方法:心力衰竭患儿34例,分成3个亚组,Ⅰ度心衰组14例,Ⅱ度心衰组13例,Ⅲ度心衰组7例。分别测定治疗前后血浆ANP水平、血清肌酸激酶同工酶(CK-MB)水平,并作超声心动图检查,观察血浆ANP水平与左室射血分数(LVEF)之间的关系。选取健康儿童20例作为对照组,以了解小儿心力衰竭时ANP、CK-MB变化情况。结果(:1)心力衰竭组ANP、CK-MB水平均明显高于正常对照组,随着心力衰竭程度的加重,血浆ANP水平逐渐增高,与心衰程度呈正相关,与LVEF呈负相关。而CK-MB与心衰程度、LVEF之间均没有相关性。(2)心功能好转后ANP、CK-MB水平均明显下降(P<0.01),LVEF明显增高(P<0.01)。结论:心力衰竭时,血浆ANP水平明显增高,并且与心衰程度、LVEF有较好的关联。     

The effects of sucrose on stability of human brain natriuretic peptide [hBNP (1–32)] and human parathyroid hormone [hPTH (1–34)]

Abstract: Although the effect of sucrose on the physical stability of proteins has been well documented, its impact on their chemical stability is largely unknown. The aim of this study was to investigate the potential effects of sucrose on the structural conformation of human brain natriuretic peptide [hBNP (1–32)] and the synthetic human parathyroid hormone [hPTH (1–34)], and link these effects to chemical degradation pathways of these peptides. The stability of hBNP (1–32) and hPTH (1–34) was studied at pH 5.5. Aggregation was monitored using size exclusion high‐performance liquid chromatography (SE‐HPLC), whereas oxidation and deamidation products were measured by reversed phase (RP) HPLC. Fourier transform infrared (FT‐IR) spectroscopy was used to study the peptides’ conformation. Sucrose retarded aggregation, deamidation, and oxidation of hBNP (1–32) and hPTH (1–34), with a maximum effect at relatively high concentrations (as much as 1 m ). FT‐IR spectroscopy indicated that sucrose maintained the native conformation of hBNP (1–32) and induced small conformation changes in the hPTH (1–34) structure. Sucrose enhanced the stability of hBNP (1–32) and hPTH (1–34) in liquid formulations. The stabilizing effect of sucrose was due to a large extent to retardation of oxidation and deamidation of hBNP (1–32) and hPTH (1–34).