Antibacterial activity of multiple antigen peptides homologous to a loop region in human lactoferrin

Abstract: An 11-residue peptide (FQWQRNMRKVR) homologous to just over half the loop region of human lactoferricin is thought to be responsible for antimicrobial properties of human lactoferricin. Multiple antigen peptides (MAP) of the 11-residue peptide exerted significant antibacterial effects against a broad spectrum of bacteria including MRSA. More than eight branching was favourable for increasing its antibacterial activity. Our report shows a novel possibility for MAP to increase the activity of antibiotic peptides other than simply to stimulate antibody production, as reported so far.     

Enhancing the antibacterial activity of PMAP‐37 by increasing its hydrophobicity

With increasing resistance against conventional antibiotics, there is an urgent need to discover novel substances to replace antibiotics. This need provides an opportunity for the development of antimicrobial peptides (AMPs). To develop new AMPs with effective and safe therapeutic effects, two PMAP‐37 analogs called PMAP‐37(R13‐I) and PMAP‐37(K20/27‐I) were designed to increase hydrophobicity. Antimicrobial susceptibility testing and animal infection models were used to assess their antibacterial activity. The results showed that the minimal inhibitory concentrations of PMAP‐37(R13‐I) were lower than those of PMAP‐37 for two gram‐negative strains. Compared with PMAP‐37, PMAP‐37(K20/27‐I) not only inhibited the growth of most bacterial strains, but also exhibited antibacterial activity against Shigella flexneri CICC21534. In addition, PMAP‐37(K20/27‐I) exhibited pH and thermal stability. PMAP‐37(R13‐I) had a therapeutic effect only in mice infected with Salmonella typhimurium SL1344. However, PMAP‐37(K20/27‐I) exhibited the therapeutic effects, whether in the clinical symptoms, the tissue lesions, or the tissue bacterial loads and the survival rates in mice infected with Staphylococcus aureus ATCC25923 or S. typhimurium SL1344. Therefore, PMAP‐37(K20/27‐I) can be used as a substitute for antibiotics against infection with bacterial strains.     

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity

Abstract: A 25‐mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure–activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an α‐helical structure in the presence of DOPC/DOPG (3 : 1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye‐leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye‐leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that α‐helicity, rather than hydrophobicity, affects the properties and activity of the peptide.     

载铜蒙脱石及其杀灭大肠杆菌机制的研究

为制备载铜蒙脱石(Cu-MMT)并研究其杀菌活性及机制，采用离子交换法制得Cu-MMT，对其结构与表面特性进行表征。以大肠杆菌为试验菌株，检测Cu-MMT对细菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)， Cu-MMT杀菌过程中菌液胞内酶活性的变化，并观察细菌形态。结果显示，载铜后蒙脱石的离子交换容量增大，但比表面积和表面负电荷密度下降；Cu-MMT对大肠杆菌的MIC和MBC分别为0.16和0.64 mg·mL^-1；Cu-MMT可使细菌细胞膜受损，胞内酶诸如冬氨酸氨基转移酶、乳酸脱氢酶和丙氨酸氨基转移酶等外泄。Cu-MMT对细菌具有较强的杀灭活性，其杀菌机制：Cu-MMT与细菌发生吸附作用，使细菌细胞膜形态和通透性改变，胞内物外泄而死亡。     

Deuterated analogues of 4,8‐dimethyldecanal,the aggregation pheromone of Tribolium castaneum: synthesis and pheromonal activity

To elucidate the deuterium isotope effect (DIE) in pheromonal activity and to investigate the biosynthetic pathway of 4,8‐dimethyldecanal (4,8‐DMD; 1 ), the aggregation pheromone of the red flour beetle (Tribolium castaneum), deuterated analogues of 4,8‐DMDs ( 2 , 3 , 4 , and 5 ), were synthesized and their pheromonal activities were tested using a two‐hole pitfall olfactometer. Although no apparent DIE was observed in their pheromonal activities, 4,8‐DMD‐1‐d₁ ( 2 ) was less attractive than other analogues, which suggested that the bond distance between the formyl group of 1 and its receptor was critical in pheromone recognition by T. castaneum. Copyright © 2004 John Wiley & Sons, Ltd.     

Modulation of neurotoxicant-induced increases in intracellular calcium by phytoestrogens differ for amyloid beta peptide (Abeta) and 1-methyl-4-phenyl-pyridine (MPP(+))

Neurotoxicant-induced elevation of intracellular calcium (Ca(2+)) and modulation by phystoestrogens were examined in vitro using human neuroblastoma SH-SY5Y cells cultured with amyloid beta-peptide (Abeta) and 1-methyl-4-phenyl-pyridine (MPP+). Although Abeta itself did not increase Ca(2+), it exacerbated the effects of carbachol. The elevation of Ca(2+) caused by the agents in combination could be reduced by pretreatment with the phytoestrogens equol and genistein, as well as by the L-type Ca(2+) channel blocker nifedipine. MPP+ exposure also elevated Ca(2+), an effect blocked by nifedipine but not by the phytoestrogens. As opposed to phytoestrogens, nifedipine was also able to significantly reduce cell death caused by higher concentrations of MPP(+) in the LDH viability assay. The results suggest that phytoestrogens are unlikely to serve as general cellular protectants for neurotoxicants with different mechanisms of action. The concentrations of Abeta and MPP(+) affecting Ca(2+) release did not inhibit cell viability as measured with the LDH release assay. This indicates that mechanisms involved with toxicity can be studied at doses that are not lethal.     

Design of a pH‐sensitive pore‐forming peptide with improved performance

Abstract: GALA is a 30 residue synthetic peptide designed to interact with membranes in a pH‐sensitive manner, with potential applications for intracellular drug and gene delivery. Upon reduction of the pH from neutral to acidic, GALA switches from random coil to α‐helix, inserts into lipid bilayers, and forms oligomeric pores of defined size. Its simple sequence and well‐characterized behavior make the peptide an excellent starting point to explore the effects of sequence on structure, pH sensitivity, and membrane affinity. We describe synthesis and characterization of two derivatives of GALA, termed GALAdel3E and YALA. GALAdel3E has a deletion of three centrally located glutamate residues from GALA, while YALA replaces one glutamate residue with the unusual amino acid 3,5‐diiodotyrosine. Both derived peptides retain pH sensitivity, showing no ability to cause leakage of an encapsulated dye from unilamellar vesicles at pH 7.4 but substantial activity at pH 5. Unlike GALA, neither peptide undergoes a conformational change upon reduction of the pH, remaining α‐helical throughout. Interestingly, the pH at which the peptides activate is shifted, with GALA becoming active at pH ～5.7, GALAdel3E at pH ～6.2, and YALA at pH ～6.7. Furthermore, the peptides GALAdel3E and YALA show improved activity compared with GALA for cholesterol‐containing membranes, with YALA retaining the greatest activity. Improved activity in the presence of cholesterol and onset of activity in the critical range between pH 6 and 7 may make these peptides useful in applications requiring intracellular delivery of macromolecules, such as gene delivery or anti‐cancer treatments.     

石榴皮鞣质的抗菌活性及其对金黄色葡萄球菌的抗菌机制

目的:研究石榴皮鞣质(tannins from Pericarpium Granati,TPG)的抗菌活性及对金黄色葡萄球菌(Staphylococcus aureus,SA)的抗菌机制。方法:实验采用牛津杯法和试管二倍稀释法研究了石榴皮鞣质对5种不同细菌的抗菌活性,并以药物敏感性最强的金黄色葡萄球菌为供试菌,通过测定菌体胞外碱性磷酸酶(AKP)含量、胞外钾离子(K⁺)浓度、大分子核酸的吸光度值、胞内和胞外总三磷酸腺苷酶(T-ATPase)、聚丙烯酰胺凝胶电泳(SDS-PAGE)和透射电镜(TEM)观察,研究了石榴皮鞣质对金黄色葡萄球菌的抗菌机制。结果:石榴皮鞣质对革兰阳性菌具有较强的抗菌活性,而对革兰阴性菌抗菌活性较弱,经药物作用后金黄色葡萄球菌菌体胞外AKP、K⁺和核酸含量与对照组相比均升高,胞内ATP酶浓度显著降低。SDS-PAGE分析表明石榴皮鞣质能够抑制菌体蛋白质合成,尤其是大相对分子质量蛋白。透射电镜表明菌体细胞膜结构和完整性被破坏。结论:石榴皮鞣质对革兰阳性菌具有较强的抗菌活性,能够显著影响金黄色葡萄球菌的细胞壁和细胞膜结构,且对菌体内蛋白合成具有一定的抑制作用。     

Synthesis,metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD₄ present on T₄ helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4–8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10^-11-10^-10M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4–8)-OH abolished the biological action. In the octapeptide series, whereas the α-amino butyric acid (Abu) substitution for Thr⁴ was well tolerated, the same “slight” structural change at Thr⁵ or Thr⁸ was very detrimental. Finally, [d -Asn⁶]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr⁴ does not interfere with biological characteristics of peptides. With regard to the enzymic degradation, the in vitro experiments showed the susceptibility of peptide T to rapid metabolism by human or rat plasma (T1/2 = 5.2 min), rat brain (T1/2 = 2.1 min) and kidney (T1/2 = 0.4 min) enzymes. The main metabolic product appears to be the C-terminal heptapeptide, which retains, in turn, a low enzymatic stability.     

三维氨基酸结构描述子矢量SVRDF及其在肽QSAR中的应用

为了建立一种新型氨基酸结构描述子并用于多肽定量构效关系研究，本研究从天然氨基酸150个radial distribution function (RDF)指数经主成分分析得出一种新3D氨基酸描述子-SVRDF(principal component scores vector of radial distribution function)，应用该描述子并通过偏最小二乘(PLS)对苦味二肽、催产素及促凝血酶原激酶抑制剂3个体系建立定量构效关系模型。对3个体系建模复相关系数(r²_cum)与交互检验复相关系数(Q²_cum)分别为0.766和0.724； 0.941和0.811； 0.996和0.919。研究结果表明，SVRDF描述子能够系统地表征肽与生物活性相关的结构信息，有望成为多肽定量构效关系研究中一种有效的结构表征方法。     

血管基膜衍生多功能肽在大肠杆菌中的表达及抗肿瘤活性的测定

目的 构建血管基膜衍生多功能肽(VBMDMP)原核表达载体、诱导表达GST-VBMDMP融合蛋白,观察其对Lewis肺癌自发转移瘤模型的影响。方法 人工合成VB-MDMP DNA序列(人源性IgG3上游铰链区连接肽连接的Tumstatin的两个功能区获得性肽基因序列),构建克隆载体PUC19-VBMDMP,亚克隆到pGEX-4T-1原核表达载体上,经测序和酶切分析鉴定获得了未发生移码突变的PGEX-4T-1-VBMDMP融合载体质粒。转化入大肠杆菌后,用 IPTG诱导表达融合蛋白。Glutathione sepharose 4B层析柱纯化蛋白表达产物。采用Lewis肺癌自发转移瘤模型,检测VBMDMP的抗肿瘤作用。结果 成功的构建pUC19-VB-MDMP克隆和pGEX-4T-1-VBMDMP表达载体,在大肠杆菌获得稳定表达,用Glutathione sepharose 4B层析柱获得纯化的GST-VBMDMP融合蛋白。VBMDMP(GST-VBM-DMP 10、6、2 mg·kg~(-1))对小鼠Lewis肺癌原发瘤具有抑制作用(瘤重抑制率分别为95.5％,80.4％,60.05％)。VBMDMP(GST-VBMDMP 10,6,2 mg·kg~(-1))对小鼠Lewis肺癌自发性肺转移具有抑制作用(自发性肺转移瘤结节抑制率分别为94.8％,86.4％,73.9％)。结论 VBM-DMP对小鼠Lewis肺癌原发瘤和肺转移具有抑制作用。     

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Sheep myeloid antimicrobial peptide of 29 amino acids (SMAP‐29) is one of the most potent antimicrobial peptides known, with a broad spectrum of activity against bacteria, fungi, and viruses. It has also been shown to prevent infections in animals. SMAP‐29 is an α‐helical cathelicidin that acts rapidly to permeabilize membranes of susceptible organisms. However, it is also cytotoxic and hemolytic to mammalian cells. In this review, all published data on inhibition constants and hemolytic activities of SMAP‐29 are brought together for the first time, including data for the peptide of 28 residues that lacks the C‐terminal glycine and that also has the new C‐terminal residue amidated. Both peptides have been called SMAP‐29 in the literature. Antimicrobial activity of SMAP‐29 variants (including ovispirins) against Gram‐positive bacteria is also comprehensively tabulated, and anomalies in the nomenclature of the ovispirins are highlighted. The secondary structure, mode of action, and structure–activity relationships of SMAP‐29 are outlined, and the properties that indicate its therapeutic potential are identified; these are its broad‐spectrum antimicrobial activity with high potency, rapid biocidal action, limited development of bacterial resistance, synthesis by recombinant technology, ability to bind lipopolysaccharide, and demonstrated protection against infection in animal models. The limitations of SMAP‐29 are its high cytotoxicity and hemolytic activity, reduced activity under physiological conditions, and limited evidence for synergistic effects in combination with conventional antibiotics. Strategies with the potential to improve the selectivity toward pathogenic microorganisms over mammalian cells have been devised by the authors and are outlined. Drug Dev Res 70: 481–498, 2009. © 2009 Commonwealth of Australia; Published 2009 Wiley‐Liss, Inc.     

蝎镇痛活性肽AS的一个二硫键对镇痛活性的影响

目的研究蝎镇痛活性肽AS中二硫键C12-C62对其生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR)分别用Ser替代蝎镇痛活性肽AS中第12位、第62位及第12位和第62位的Cys,从而构建其3个突变体。采用金属离子螯合亲和层析方法对表达突变体蛋白进行纯化。采用小鼠扭体法测定3个突变体的镇痛活性。结果构建了3个突变体质粒,在BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析方法获得了电泳纯样品。其镇痛活性明显低于Bmk AS(P<0.01)。结论3个突变体与rBmK AS镇痛活性有明显差异,二硫键C12-C62与蝎毒镇痛活性密切相关。     

1-烷基-6-氟-1,4-二氢-7-(4-酰基-1-哌嗪基)-4-氧代喹啉-3-羧酸类似物的合成及其抗菌活性

目的 设计合成具有广谱抗菌活性的含氟喹诺酮类化合物。方法 利用吡酮酸7位哌嗪基4位氮上存在的活性氢为反应修饰基点，与磺酰氯，酰氯，氯甲酸酯进行Schotten-Baumann酰基化反应设计合成一系列含有磺酰基、酰基和烷氧羰基类氟喹诺酮类似物。结果 合成了20个含有磺酰基、酰基和氧羰基的氟喹诺酮类似物，利用元素分析、核磁共振进行了结构确认。结论 合成了16个未见报道的新化合物，初步体外活性测试结果表明，其中的4个化合物有较高的生物活性。     

Influence of the hydrophilic face on the folding ability and stability of α‐helix bundles: relevance to the peptide catalytic activity

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self‐aggregating 18‐mer peptides designed previously to adopt a high level of α‐helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four α‐helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far‐ and near‐UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well‐defined tertiary structure acting as true catalysts.     

Reaction of [18F]4‐fluorobenzenediazonium cations with cysteine or the cysteinyl group: preparation of 18F‐labeled S‐aryl‐cysteine and a radiolabeled peptide

A reaction route for the preparation of no‐carrier‐added (n.c.a.) [¹⁸F]S‐4‐fluorophenylcysteine 7 via the [¹⁸F]‐4‐fluorobenzenediazonium ion 4 is described. The key step in this radiosynthesis is the reaction of 4 with cysteine forming [¹⁸F]4‐fluorophenyldiazocysteine 6 , which is subsequently converted into 7 by irradiation with 366 nm light. 4 was synthesized by reacting 1,4‐dinitrobenzene 1 with [¹⁸F]‐fluoride in acetonitrile in a PEEK‐capillary in a microwave oven. After dilution of the reaction mixture with methanol, the resulting [¹⁸F]4‐fluoro‐1‐nitrobenzene 2 was submitted to reduction by means of H₂ with Pd on C catalyst. The resulting [¹⁸F]4‐fluoroaniline 3 was purified by HPLC and diazotized to 4 . The preparation of 4 was optimized with regard to yield and purity. The radiochemical yield of 6 was >90% (based on 3 ) while after UV irradiation and HPLC purification 45% of 7 (based on 3 ) was obtained (yields corrected for decay). The suitability of this method for labeling peptides with fluorine‐18 was demonstrated by application to the tripeptide, glutathione (GSH; γ‐L‐glutamyl‐L‐cysteinyglycine) 8 . Copyright © 2002 John Wiley & Sons, Ltd.     

Crossover assessment of serum bactericidal activity of grepafloxacin, ofloxacin and clarithromycin against respiratory pathogens after oral administration to healthy volunteers

Serum bactericidal activity was studied in a crossover manner in 10 volunteers, after 2-day administration of grepafloxacin 600 mg qd, ofloxacin 400 mg bid and clarithromycin 500 mg bid. Bactericidal activity against clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Klebsiella pneumoniae, was estimated using a standardized microdilution method. Grepafloxacin was highly active against Gram-negative organisms and adequate against pneumococci (mean, 1:13.3). Clarithromycin was very active against both penicillin-susceptible and penicillin-partially-resistant S. pneumoniae (1:102.5) but had poor activity against H. influenzae (1:3.1). Minor adverse effects were commonly associated with grepafloxacin.     

普萘洛尔治疗门脉高压性消化道出血B型尿钠肽前体水平监测及意义

目的 观察普萘洛尔治疗肝硬化门脉高压性消化道出血患者血浆B型尿钠肽前体(pro-BNP)水平.方法 随机将患者分为治疗组22例和对照组21例;治疗组给予普萘洛尔10 mg口服,每天3次;对照组不用普萘洛尔治疗,输血、抑酸等治疗相同.测定两组患者出血前和出血后以及治疗后24h、1周、2周、4周、8周B型尿钠肽前体水平.结果 治疗组和对照组患者血浆pro-BNP水平在消化道出血前明显高于消化道出血后(P＜0.01);普萘洛尔治疗后血浆pro-BNP水平降低(P＜ 0.05),2个月内再次出血发生率减低(P＜0.05),对照组血浆pro-BNP水平升高,2个月内出血发生率增加(P＜0.05).结论 普萘洛尔治疗可降低肝硬化患者血浆pro-BNP水平,延长再出血时间和减低再次出血发生率,pro-BNP水平与有效循环血容量相关,血浆pro-BNP水平可视为门脉高压性消化道出血独立预测因素.     

Antifungal activity of human salivary mucin‐derived peptide,MUC7 12‐mer,in a murine model of oral candidiasis

Abstract: Previous studies have shown that peptides derived from the N‐terminal region of the low molecular mass human salivary mucin, MUC7, possess potent in vitro cidal activity against Candida albicans and other medically important fungi. MUC7 12‐mer (residues 40–51 of the parent MUC7) peptide, having the optimal size and a net charge of +6, was found to be anticandidal in human saliva (clarified and unclarified), and its candidacidal potency was found to be superior to that of histatin 5 12‐mer (Hsn5 12‐mer). We have, therefore, explored the candidacidal potency of MUC7 12‐mer (l and d isomers) and Hsn5 12‐mer peptides in vivo. In vitro killing assay was performed to establish killing activity of the peptides against C. albicans prior to in vivo experiments. A murine model of oral candidiasis that has the characteristics of oral thrush in humans was employed for the in vivo studies, based on a previous protocol. Upon candidal induction, antifungal treatment application using agents emulsified in Pluronic F127 was performed for six consecutive days. Amphotericin B and clotrimazole emulsified in the same delivery system were used as positive control drugs. Candidacidal efficacy was evaluated microbiologically and histopathologically. Results demonstrated a considerable reduction of fungal burden by the MUC7 12‐mer peptides (l and d ), comparable to control drugs, and this effect was statistically significant, unlike the effect seen with Hsn5 12‐mer. Murine oral candidiasis model employed in this study is suitable to test the candidacidal agents employing Pluronic F127. In conclusion, MUC7 12‐mer appears to be a promising candidate as an antifungal agent for oral candidiasis.