提高噬菌体裂解酶抗菌活性的研究进展

随着细菌耐药性问题的日益严重,人们开始寻求新型抗菌制剂。噬菌体裂解酶是一种由ds DNA噬菌体编码的水解酶,能高效特异性地裂解细菌细胞壁且不易使细菌产生耐药性。由于天然裂解酶具有宿主谱窄,不能裂解革兰阴性菌等缺点,研究者对裂解酶进行了大量的设计改造。本研究主要对提高噬菌体裂解酶抗菌活性的研究进展进行综述。     

核酸转染技术和阳离子脂质体

阳离子脂质体介导转染是一种具创新性的、高效的转染技术,并且逐渐成为介导核酸进入真核细胞的标准方法。因此,如果在使用其他的转染手段并不理想时,宜试用阳离子脂质体介导的转染技术。     

D-甘露糖修饰黄芩苷阳离子脂质体的制备及其对肺癌A549细胞增殖的抑制作用

目的:制备一种D-甘露糖修饰的黄芩苷阳离子脂质体[Baicalin cationic liposome,BC-Lipo(+)],并考察其对肺癌A549细胞增殖的抑制效果。方法:用乙醇注入法制备BC-Lipo(+),并考察其药剂学性质;以肺癌A549细胞为模型,采用MTT法考察其对肿瘤细胞的抑制效果。结果:透射电镜下可见BC-Lipo(+)呈圆形或类圆形,平均粒径(111.3±2.7)nm,Zeta电位(9.6±0.3)m V,包封率(95.4±0.8)%;体外释药行为符合Ritger-Peppas方程(lnQ=0.3497lnt-1.6611,r=0.9924);A549细胞的增殖抑制率可达(88.3±5.7)%。结论:处方和制备工艺合理,BC-Lipo(+)包封率较高,粒径分布均匀,带一定的正电荷,具有明显的体外缓释特性,抑制肺癌A549细胞增殖效果显著,为深入研究肺靶向BC纳米脂质体制剂奠定了基础。     

阳离子脂质体在转基因畜禽中的应用

脂质体是由可生物降解的磷脂组成的双分子层结构,与生物膜有较大的相似性和组织相容性.阳离子脂质体作为一种新型的基因转移载体,以其生产简便、毒性低、无感染危险等优点在转基因领域越来越受到研究人员的青睐.随着转基因动物研究的不断深入,阳离子脂质体更是成为当今各种转基因方法中首选的非病毒类外源基因载体,在保护外源基因和提高其转染效率方面都发挥着重要作用.就阳离子脂质体的结构形式、介导基因转移的机制及其目前在转基因畜禽领域中的研究进展做一综述.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

用于转基因的阳离子脂质体

通过直接导入外源基因来治疗人类疾病的方法需要一种有效、安全并且可重复进行的载体,阳离子脂质体是基本满足这些条件的有限几种载体中的一员. 目前已经有十几种阳离子脂质体. 这些脂质体通过外周的电荷与DNA相结合,静电吸引形成复合物在与细胞膜相互作用后,通过细胞的内吞或融合作用使复合物进入细胞内,从而将外源基因导入细胞,这种DNA传递技术的有效性和安全性已经确立. 二例利用阳离子脂质体的人体基因治疗临床试验也已开始实施,将来会有更多的临床试验得到开展,阳离子脂质体在基因治疗领域有较好的前景.     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

噬菌体裂解酶的抗菌特性

摘要：噬菌体裂解酶是一类细胞壁水解酶,可水解肽聚糖,造成细菌的破裂。裂解酶一般具有两到三个结构域,参与对底物的催化和结合。作为一种新型的杀菌制剂,裂解酶已被越来越多地应用于化脓链球菌、肺炎链球菌、金黄色葡萄球菌等革兰氏阳性细菌病的治疗。与抗生素治疗相比,裂解酶不易使细菌产生抗性且作用相对专一,这可能是解决现在日趋严重的细菌耐药性的一种可行方法。另外,裂解酶还具有高效性,作用协同性,且自身抗体不削弱其作用等优势,使之成为未来预防、控制致病菌一种可能的新途径。     

胆固醇嵌入式阳离子脂质体运载基因研究

阳离子脂质体是一种有临床应用潜力的抗肿瘤药物递药系统,助类脂能起到稳定双层膜和降低阳性成分毒性的作用,同时提供阳性类脂的细胞渗透功能。为了进一步发掘助类脂的应用潜力,该文采用胆固醇(cholesterol)作为助类脂制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为100~140 nm,Zeta电位为45~60 mV。脂质体分别与绿色荧光蛋白基因(pGFP-N2)、荧光素酶基因(pGL3)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2),考察了其转染效率和细胞毒性。结果表明,阳离子类脂与胆固醇以1:1、1:2和1:4摩尔比例混合制备脂质体均能高效转染Hep-2细胞。毒性实验显示,阳离子类脂单独存在时对癌细胞具有一定的细胞毒性,随着胆固醇的加入,脂质体对细胞的毒性明显减小,与商品试剂DOTAP和Lipofectamine 2000相当。     

Effect of onion-type multilamellar liposomes on Trametes versicolor laccase activity and stability

Trametes versicolor laccase was encapsulated into onion-type, lipid-based multilamellar vesicles (MLVs). When encapsulated, laccase was isolated from the assay medium but was still active once freed from its capsule. The encapsulation efficiency was larger than 65% at 25 °C and 37 °C and decreased to 55% by introducing 140 mM NaCl into the buffered medium (pH = 4.5). MLVs were shown to drastically improve both laccase stability and activity. At 25 °C, laccase activity was doubled in the presence of MLVs. At 37 °C in the salt-free medium, the half-life time of laccase was increased from 2hr 30-65 h without and with MLVs, respectively. This effect was even more pronounced in the salted medium where laccase activity was unchanged for 6 days in the presence of MLVs. These beneficial effects were attributed to the immobilization of laccase onto MLV surface. Laccase activity as well as stability was notably shown to be directly correlated to MLV stability.     

羟基酪醇抑菌活性及抑菌稳定性研究

本研究探究了羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌等四种供试菌的抑菌活性及抑菌稳定性。采用试管半倍稀释法确定MIC和MBC,并探讨羟基酪醇对供试菌的生长和细胞膜完整性的影响以及在不同介质下的抑菌稳定性。结果表明,羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌的MIC分别为0.625、0.625、1.250、2.500 mg/mL,MBC分别为1.250、1.250、2.500、5.000 mg/mL。与对照组相比,四种供试菌核酸和可溶性蛋白泄漏显著,细胞膜的完整性被破坏。在不同NaCl浓度下,羟基酪醇对枯草芽孢杆菌的抑菌活性稳定;在1.0%和2.0%NaCl浓度下,羟基酪醇对大肠杆菌和铜绿假单胞杆菌的抑菌活性稳定;在2.0%NaCl介质下低浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定,在0.5%、1.5%和2.0%NaCl介质下高浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定。在蔗糖介质中,羟基酪醇对四种供试菌的抑菌活性均不稳定。因此,羟基酪醇可以作为一种新型的防腐剂。     

Peroxidase biocatalysis in water-soluble ionic liquids: activity,kinetic and thermal stability

Abstract

The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [C_xmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C₄mim][Cl] and [C₄mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C₄mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.     

Potential dopamine prodrug-loaded liposomes: preparation,characterization, and in vitro stability studies

Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of the blood-brain barrier (BBB). Condensation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the BBB endogenous transporters and easily enter the brain. To improve the bioavailability of the dopamine prodrug, 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DOPH), it was encapsulated in unilamellar liposomes of dimiristoylphosphatidylcholine (DMPC) and cholesterol. Vesicles were characterized by dynamic light scattering in order to evaluate their dimensions and vesicle stability, by zeta-potential measurements, by means of electronic microscopy after freeze-fracture and differential scanning calorimetry. The influence of vesicle composition on DOPH chemical and enzymatic stability was also evaluated. The formulated liposome suspensions were found to be stable, monodisperse systems with a negative zeta potential. From the obtained results, it is possible to conclude that, in studied samples, DOPH inclusion in liposomes offers the possibility of preventing photodegradation and of enhancing in vitro plasma stability. These studies suggest the potential of these formulations as a method to prevent DOPH chemical degradation and enzymatic metabolism.     

噬菌体粉剂的制备及抗逆性与稳定性

【背景】细菌耐药已成为全球严重的公共卫生问题,养殖业是细菌耐药性产生的重要源头之一。我国正在全力推进"减抗、限抗、禁抗"战略。新型安全高效的抗生素替代品成为当前养殖业的重要需求。噬菌体因其能有效裂解细菌被认为是一个重要的突破口,但噬菌体作为活体微生物,在保存和使用时存在稳定性差、利用率低等问题。【目的】制备噬菌体粉剂,提高噬菌体的抗逆性和稳定性,为噬菌体在养殖业中的应用提供技术支持。【方法】选用大肠杆菌噬菌体BpEP4,采用嵌段式聚醚F-68包被噬菌体,然后负载于脱脂米糠制得噬菌体粉剂,双层琼脂平板法测定粉剂的噬菌体效价,研究其耐高温性能、pH稳定性与常温保存稳定性。【结果】噬菌体粉剂可以在100℃保持噬菌体的活性,pH耐受范围为2.0-12.0。常温下保存3个月噬菌体效价无明显降低。【结论】粉剂显著提高了噬菌体的抗逆性与稳定性,具有很好的应用价值和推广前景。     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, α-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (a_W) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and α-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter α, determined from the activity coefficient of water, representing the deviation of a_W from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and α-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant a_W. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

Construction and evaluation in vitro and in vivo of tedizolid phosphate loaded cationic liposomes

Abstract

First, the SA-TDZA-Lips were prepared by reverse-phase evaporation method. Then, the drug release behaviour was evaluated by dynamic membrane dialysis in vitro and the preliminary safety was evaluated by haemolysis method. Finally, with tedizolid phosphate injection (TDZA-Inj) and tedizolid phosphate loaded liposomes (TDZA-Lips) as the control groups, the pharmacokinetic characteristic and tissues distribution of SA-TDZA-Lips were evaluated after intravenous injection. As a result, the stearylamine modified tedizolid phosphate liposomal delivery system was constructed successfully and the particle size was 194.9?±?2.93?nm. The encapsulation efficiency (EE) was 53.52?±?2.18%. The in vitro release of SA-TDZA-Lips was in accordance with Weibull equation. And there was no haemolysis happened, which indicated good preliminary safety for injection. The results of pharmacokinetics showed that the t1/2β increased by 0.74 times and 0.51 times higher than that of TDZA-Inj group and TDZA-Lips group, respectively. The MRT of SA-TDZA-Lips was 1.30 and 1.09 times higher than that of TDZA-Inj group and TDZA-Lips group, respectively. The AUC was 2.40 times and 0.23 times higher than that of TDZA-Inj group and TDZA-Lips group, respectively. The tissue distribution results showed that the relative uptake rate (Re) of TDZA in the lung was 1.527, which indicated the targeting. In conclusion, the SA-TDZA-Lips prepared in this study had several advantages like positive charge, strong cell affinity, prolonged circulation time in vivo, sustained release effect, and increased drug concentration in lungs. All advantages above provided significant clinical value of application for the treatment of bacterial pneumonia with tedizolid phosphate.     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。     

Iron(III) complexes: Preparation,characterization, antibacterial activity and DNA-binding

Iron(III) have been combined to well known quinolones (ciprofloxacin) and some Schiff bases with the help of coordination approach. Characterization of these compounds have been done using elemental analysis, magnetic measurements, thermogravimetric analysis, IR, UV-VIS, ¹H NMR and ¹³C NMR spectral investigation. Analytical studies suggest that the iron(III)-quinolone complexes assume a six-coordinated dimeric distorted octahedral geometry. All the compounds show a good antibacterial activity against broad range of bacteria like Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Serratia marcescens, whereas no significant inhibition towards growth of fungal strains like Aspergillus Niger, Aspergillus flavus and Lasiodiplodia theobromae. Analyses of all these compounds show effective sperm herring DNA inhibition.