蜂毒溶血肽(Melittin) cDNA的克隆及序列分析

为了克隆得到正确序列的蜂毒溶血肽cDNA，对蜂毒中的蜂毒溶血肽进行了研究。实验从蜜蜂毒腺中提取总RNA，通过RT－PCR方法扩增到了蜂毒溶血肽的cDNA，扩增产物克隆到pT7Blu-T载体，转化大肠杆菌JM109，DNA序列分析结果表明克隆得到的蜂毒溶血肽cDNA脱氧核糖核苷酸序列正确。     

用粗粒化分子动力学方法研究蜂毒素与磷脂双分子层的相互作用

为了了解蜂毒素的生物活性机理,采用粗粒化分子动力学模拟方法研究了蜂毒素在水环境中的性质以及与多种磷脂双分子层膜的相互作用.结果表明:无论是在水环境还是在磷脂双分子层中,蜂毒素都能形成聚集体.在每个聚集体中,蜂毒素分子之间的相对位置不断变化,会在蜂毒素分子之间形成无特定形状的空隙,从而在膜内产生暂时的孔洞或者跨膜通道.蜂毒素分子的N端和C端带正电荷的残基与磷脂膜带负电荷的磷脂头之间的静电相互作用是蜂毒素发挥功能的关键因素.     

Design of high payload PLGA nanoparticles containing melittin/sodium dodecyl sulfate complex by the hydrophobic ion-pairing technique

The water-soluble peptide, melittin, was modified with an anionic agent, sodium dodecyl sulfate by hydrophobic ion-pairing. Investigations showed that the formed complex was very soluble in organic solvent, especially, in dimethylsulfoxide and dehydrated alcohol. Furthermore, the physiochemical properties of the complex in the solid state or in an aqueous medium were characterized using octanol/water partition measurement, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The complex was formulated into poly(d,l-lactide–co-glycolide acid) nanoparticles by an emulsion solvent diffusion method. It was found that the nanoparticles of about 130 nm in size can be produced with a high encapsulation efficiency, and the entrapment of nanoparticles prepared with the formed complex increased from about 50% to nearly 100% compared with that for pure melittin. Moreover, the growth inhibitory effects of modified melittin and melittin-loaded nanoparticles in breast cancer MCF-7 cells were not changed comparing with free melittin as determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay.     

Smart aptamer-modified calcium carbonate nanoparticles for controlled release and targeted delivery of epirubicin and melittin into cancer cells in vitro and in vivo

To explore the effect of combination therapy of epirubicin (Epi) and melittin (Mel) to cancer cells, calcium carbonate nanoparticles (CCN), as carriers, were developed which were modified with MUC1-Dimer aptamers as targeting agents. Both Epi and Mel were delivered at the same time to cancer cells overexpressing the target of MUC1 aptamer, mucin 1 glycoproteins (MCF7 and C26 cells). CCN were prepared with a water-in-oil emulsion method. Epi and Mel were separately encapsulated in CCN and the nanoparticles were modified with MUC1-Dimer aptamers. In vitro studies, including MTT assay, flow cytometry analysis and fluorescence imaging were applied to investigate the targeting and cell proliferation inhibition capabilities of MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex in the target (MCF-7 and C26 cells) and nontarget (HepG2) cells. Also, the function of the developed complexes was analyzed using in vivo tumor growth inhibition. The release of Epi from MUC1-Dimer aptamer-CCN-Epi complex was pH-sensitive. Cellular uptake studies showed more internalization of the MUC1-Dimer aptamer-CCN-Epi complex into MCF-7 and C26 cells (target) compared to HepG2 cells (nontarget). Interestingly, the MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex indicated very low toxicity as compared to target cells. Moreover, co-delivery of Epi and Mel using the mixture of MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex exhibited strong synergistic cytotoxicity in MCF-7 and C26 cells. Furthermore, the presented complexes had a better function to control tumor growth in vivo compared to free Epi.     

Intestinal Drug Absorption Enhancers: Synergistic Effects of Combinations

Although many absorption enhancers have been investigated, very few are used clinically. A need exists therefore for more effective absorption enhancers. The drug-absorption-enhancing effects of combinations of N-trimethyl chitosan chloride (TMC) with degrees of quaternization of 48 and 64%, dicarboxymethyl chitosan oligosaccharide, and chitosan lactate oligomer with monocaprin and melittin were compared to their individual performances using the in vitro Caco-2 cell model. Combining the absorption enhancers showed synergism in both the reduction of the transepithelial electrical resistance (TEER) and the enhancement of the transport of a macromolecular model compound across this intestinal epithelial cell layer. Lower concentrations of the absorption enhancers in the combination groups exhibited greater effects on the epithelial cells compared with the individual absorption enhancers.     

Targeted delivery of melittin to cancer cells by AS1411 anti-nucleolin aptamer

Melittin, a small water-soluble cationic amphipathic α-helical linear peptide, consisted of 26 amino acids, is the honeybee venom major constituent. Several reports have proved the lytic and apoptotic effects of melittin in several cancerous cell lines. In this study, we aimed to fabricate an AS1411 aptamer–melittin to specifically deliver melittin to nucleolin positive cells (A549). Melittin was covalently attached to antinucleolin aptamer (AS1411) and its toxicity in A549 (nucleolin positive) and L929 (nucleolin negative) was studied using MTT and Annexin V flow cytometry methods. Aptamer–melittin conjugate formation was confirmed by gel electrophoresis. Hemolytic effect of aptamer–melittin conjugate was compared to melittin alone. The aptamer–melittin conjugate showed efficient cell uptake and was more cytotoxic in A549 cells than melittin (p?melittin complex delivery occurred through receptor-ligand interaction on the cell surface. Moreover, aptamer–melittin showed a significantly less hemolytic activity as compared with free melittin. This study showed that melittin could be specifically delivered to A549 cells when it was covalently conjugated to antinucleolin aptamer (AS1411) in vitro. This system can reduce the cytotoxic effects of melittin on cells with no nucleolin receptor overexpression which comprise most of normal cells such as L929 cells.     

Bee Venom Melittin Disintegrates the Respiration of Mitochondria in Healthy Cells and Lymphoblasts,and Induces the Formation of Non-Bilayer Structures in Model Inner Mitochondrial Membranes

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O₂ consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O₂ consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin’s cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by ³¹P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.     

蜂毒肽的分离纯化及结构鉴定

目的 对蜂毒肽进行分离纯化,并进行结构鉴定。方法 以意大利蜜蜂蜂毒为原料,经溶解、超声、浸提、过滤等,并制备C18液相色谱柱,对一次纯化采用的流动相、洗脱程序进行优化,对二次纯化时进样量、进样浓度进行选择,冻干后得到高纯度蜂毒肽。利用基质辅助激光解析电离飞行时间质谱法(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)和液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)进行分子量测定等定性研究,利用傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FT-IR)进行二级结构表征,尝试利用低场核磁共振(nuclear magnetic resonance, NMR)技术对其氢谱定性解析,基于高效液相色谱法(high performance liquid chromatography, HPLC)的面积归一化法测定蜂毒肽纯度。结果...     

Assessment of antimicrobial activity of melittin encapsulated in bicontinuous microemulsions prepared using renewable oils

The objective of this study is to demonstrate that melittin, a well-studied antimicrobial peptide (AMP), can be solubilized in an active form in bicontinuous microemulsions (BMEs) that employ biocompatible oils. The systems investigated consisted of Winsor-III and -IV BME phases composed of Water/Aerosol-OT (AOT)/Polysorbate 85/isopropyl myristate and a Winsor-IV BME employing Polysorbate 80 and limonene. We found that melittin resided in an α-helix-rich configuration and was in an apolar environment for the AOT/Polysorbate 85 Winsor-III system, suggesting that melittin interacted with the surfactant monolayer and was in an active conformation. An apolar environment was also detected for melittin in the two Winsor-IV systems, but to a lesser extent than the Winsor-III system. Small-angle X-ray scattering analysis indicated that melittin at a concentration of 1.0 g/L_aq in the aqueous subphase of the Winsor-IV systems led to the greatest impact on the BME structure (e.g., decrease of quasi-periodic repeat distance and correlation length and induction of interfacial fluidity). The antimicrobial activity of the Polysorbate 80 Winsor-IV system was evaluated against several bacteria prominent in chronic wounds and surgical site infections (SSIs). Melittin-free BMEs inhibited the growth of all tested bacteria due to its oil, limonene, while the inclusion of 1.0 g/L_aq of melittin in the BMEs enhanced the activity against several bacteria. A further increase of melittin concentration in the BMEs had no further enhancement. These results demonstrate the potential utility of BMEs as a delivery platform for AMPs and other hydrophilic and lipophilic drugs to inhibit antibiotic-resistant microorganisms in chronic wounds and SSIs.