抗菌肽buforinⅡ的研究进展

抗菌肽buforinⅡ是由亚洲蟾蜍bufo bufo gargarizans胃组织中分离得到一种烈性抗菌肽,具有很强的抗菌活性。它的结构和多数α螺旋抗菌肽相似,但作用机制却有较大的区别。BuforinⅡ利用其特有的结构在不造成膜穿孔的情况下迅速通过细胞膜,与胞内的生物大分子核酸结合,诱发细胞死亡。本文就buforinⅡ的结构,作用机制,研究现状及其应用前景进行阐述。     

Buforin Ⅱ衍生肽的分子设计、结构分析及活性研究

在Buforin Ⅱ衍生肽BF2-A的结构特征基础上,重新设计新抗菌肽BF2-X。BF2-X是将BF2-A的N-端保持不变,在第10位精氨酸上接入一个3次重复的α-螺旋序列RLLR,并将第8位上的缬氨酸用亮氨酸取代。生物信息学分析表明,与BF2-A相比,BF2-X的正电荷增加,疏水性比例提高,螺旋度增加,同时两亲性也增强。化学合成后经圆二色谱检测表明,BF2-A/X在水相中是一种无规则卷曲结构,当置于模拟细胞膜疏水环境的50%三氟乙醇中时,BF2-A/X会被诱导成α-螺旋结构,并且BF2-X的α-螺旋含量明显高于BF2-A。抑菌试验表明BF2-X对细菌以及真菌都具有广谱抗菌活性,BF2-X对细菌的抗菌活性要比BF2-A强。BF2-X的α-螺旋含量的提高可能与抗菌活性的增强有直接的关系。BF2-A/X对小鼠红血球不具有体外溶血活性。     

金黄色葡萄球菌肠毒素A基因全序列的克隆、鉴定与扩增

目的 克隆SEA基因作为后续基因治疗的目的基因。方法 以金黄色葡萄球茵基因组DNA(ATCC-13565)为模板,以金黄色葡萄球菌肠毒素A(SEA)基因特异性引物为介导，采用普通PCR和降落PCR方法分别克隆SEA基因全序列，克隆入T载体后进行DNA测序。结果 2％琼脂糖凝脂电泳和DNA测序证实该基因克隆成功。结论 通过PCR方法，可以获得后续实验所需的SEA基因。     

抗菌肽对耐甲氧西林金黄色葡萄球菌的抗菌机制研究进展

目的:了解抗菌肽对耐甲氧西林金黄色葡萄球菌(MRSA)的抗菌机制,以期为抗菌肽临床治疗MRSA肺炎提供参考。方法:查阅近年来国内外相关文献,就各种抗菌肽对MRSA的抗菌机制的相关研究进行归纳和总结。结果与结论:抗菌肽相对于抗菌药物拥有较多优势——(1)抗菌肽是生物天然免疫系统的组成部分,容易获得;抗菌肽氨基酸数较少、肽链较短,减小了合成抗菌肽的难度,为大量人工合成抗菌肽提供了可能性。(2)抗菌肽表面富含正电荷,YD1、Melittin和Bac8c均通过其表面的正电荷与MRSA表面的负电荷结合并黏附于细菌表面,进一步破坏细胞膜从而杀灭细菌;LL-37能抑制MRSA生物膜的形成并破坏已经形成的MRSA生物膜;h BD3-CBD通过在MRSA周围聚集进而发挥杀菌作用;J-AA、J-RR和J-AR利用其结构特殊性,通过内/外膜透化机制,破坏MRSA细胞膜,从而杀伤细菌。上述机制皆不涉及受体与配体之间的结合,避免了MRSA对抗菌肽产生耐药性。(3)大部分抗菌肽在极低的MIC下即已对MRSA展示出了强大的杀菌作用。抗菌肽的使用也存在一定的局限性——(1)抗菌肽的肽链较短,增加了提取难度,人工合成抗菌肽则提高了药物成本。(2)抗菌肽的短肽链和简单结构,使其稳定性较差。(3)抗菌肽是一种异种蛋白,可能诱发患者产生一系列的免疫反应和毒性作用。     

2-羧甲氧基苯甲醛苯甲酰腙及其配合物抑菌活性的构效关系

目的 :研究 2 羧甲氧基苯甲醛苯甲酰腙及其配合物的抑菌活性。方法 :合成配体 2 羧甲氧基苯甲醛苯甲酰腙及其配合物 ,采用K B法检测其对金黄色葡萄球菌、大肠埃希菌和枯草芽胞杆菌的生长抑制作用。结果 :配体 2 羧甲氧基苯甲醛苯甲酰腙仅抑制金黄色葡萄球菌的生长 ,其配合物能抑制金黄色葡萄球菌、大肠埃希菌和枯草芽胞杆菌的生长 ,形成抑菌环 ,尤其Cu配合物形成抑菌环直径最大。结论 :2 羧甲氧基苯甲醛苯甲酰腙与金属、稀土形成的配合物均有一定的抑菌作用 ,Cu配合物抑菌活性最强     

合成棕点湍蛙抗菌肽的抗菌活性评价

目的对合成棕点湍蛙抗菌肽的抗菌作用进行分析,探讨合成产物与天然产物的一致性以及临床应用价值。方法体外抗菌试验测定合成抗菌肽及与庆大霉素、青霉素和链霉素联合应用对金色葡萄球菌和大肠埃希菌的最小杀菌浓度(MBC)。结果合成抗菌肽对金黄色葡萄球菌和大肠埃希菌均有一定的杀菌作用,MBC值分别为200 mg.L-1和100 mg.L-1,但活性低于生物化学方法提取的天然产物。合成抗菌肽与青霉素、链霉素联合使用后抗菌作用有增强趋势,其中与青霉素联合使用的效果较明显,MBC值达到青霉素/合成肽为0.625 mg.L-1/25 mg.L-1。结论抗菌肽的联合应用可能有助于改善病原性细菌对传统抗生素的耐药性。     

重组人ⅡA型磷脂酶A2对金黄色葡萄球菌超微结构的影响

摘 要 目的：观察重组人ⅡA型磷脂酶A2（PLA2）对金黄色葡萄球菌超微结构的影响。方法: 用琼脂平板计数法测定重组人ⅡA型PLA₂对金黄色葡萄球菌的杀菌活性,ⅡA型PLA₂与金黄色葡萄球菌共同孵育在RPMI-1640培养液中,37℃水浴,分别孵育30,60,120 min后,应用透射电镜观察细胞超微结构的变化。结果:重组人ⅡA型PLA₂杀灭99%金黄色葡萄球菌的最低浓度（MBC）为80 ng·ml^-1。ⅡA型PLA₂作用30～60 min后,金黄色葡萄球菌的菌体略显肿胀,细胞壁与细胞膜因局部分离而出现空隙,细胞膜、细胞壁在横隔结构处裂开。当ⅡA型PLA₂作用120 min后,可见细菌的细胞壁破坏严重,胞质外流。结论:ⅡA型PLA₂对金黄色葡萄球菌有较强的抗菌作用,并可能通过破坏细菌的细胞壁或细胞膜结构来抑制细菌的生长。     

Losartan和CGRP对血管紧张素Ⅱ诱导血管平滑肌细胞增殖作用的影响

目的比较非肽类血管紧张素Ⅱ受体Ⅰ型阻断剂氯沙坦(Losartan)和血管活性肽降钙素基因相关肽(CGRP)对血管紧张素Ⅱ诱导的血管平滑肌细胞增殖作用的影响,为探讨此两类物质的降压机制提供实验依据。方法采用MTT,3H-参入法和流式细胞仪分别测定血管紧张素Ⅱ刺激下,Losartan或CGRP干预下血管平滑肌细胞的增殖变化,W est-ern b loting法测定不同状态下血管平滑肌细胞内ERK1/2的活性变化。结果Losartan或CGRP能抑制血管紧张素Ⅱ刺激下血管平滑肌细胞的生存率、DNA合成、细胞周期增殖指数,以及细胞内ERK1/2的活性,并呈剂量依赖性。而且,CGRP抑制作用强于Losartan。结论Losartan或CGRP能抑制血管紧张素Ⅱ刺激下血管平滑肌细胞增殖,其细胞内的信号传导途径与ERK1/2有关。     

甲氧西林金黄色葡萄球菌的耐药基因与耐药性关系

金黄色葡萄球菌对甲氧西林耐药的主要机制之一，是细菌染色体上的一段外源DNA（mecA基因）编码产生对青霉素亲和力极低的青霉素结合蛋白2（PBP2a），mecA基因在葡萄球菌属中分布广泛，它和细菌染色体上的一些辅助基因和调控基因的共同作用下影响细菌胞壁合成，使细胞表现出异质性耐药。而且MRSA是医院内和社区感染的重要致病菌，由于感染后治疗困难，因此成为国内外学者关注与研究的热点。本文主要从mecA基因的来源、传播、结构功能、调控基因和辅助基因（fem或aux基因）等方面进行综述。     

丁香酚对压疮致病菌金黄色葡萄球菌及其耐药菌的抑菌作用及机制

目的 明确丁香酚对压疮致病菌金黄色葡萄球菌Staphylococcus aureus的体外抑菌作用及机制。方法 采用微量肉汤稀释法和和生长曲线确定丁香酚对金黄色葡萄球菌的抑菌活性；通过碱性磷酸酶（AKP）活性、细菌核酸蛋白泄漏的检测，细菌超微结构的扫描和透射电镜观察以及菌体对碘化丙啶（PI）和N-菲酰-次巯基-L-丙氨酸（NPN）的摄取来探讨丁香酚处理后金黄色葡萄球菌细胞膜的通透性和完整性；以结晶紫染色法和实时荧光定量PCR法研究丁香酚对菌体生物被膜（BF）生成、成熟以及菌体黏附、侵袭相关毒力因子表达水平的影响。结果 丁香酚对金黄色葡萄球菌标准菌和耐甲氧西林金葡菌（MRSA）的最低抑菌浓度（MIC）分别为0.25、0.125 mg/mL。丁香酚处理后可以提高细菌培养液中核酸、蛋白浓度及AKP的活性；菌体出现皱缩和破裂，菌体内膜和外膜中分别出现PI和NPN荧光，菌体结构破坏和荧光强度与丁香酚浓度呈正相关。丁香酚能够抑制金黄色葡萄球菌菌株BF的生成，且对成熟BF具有明显清除作用，显著降低BF生成相关基因agrA、sarA、cidA和icaA及黏附因子clfA、clfB、fnbA和fnbB的基因表达水平；与标准菌株相比，丁香酚对MRSA的作用更明显。结论 丁香酚对金黄色葡萄球菌标准菌株和耐药菌株均具有显著抗菌作用，可破坏菌体细胞结构，改变细胞膜通透性和完整性，抑制BF的生成，并对成熟BF具有清除作用，抑制细菌BF及黏附侵袭相关毒力因子的转录和表达，可能是艾灸促进压疮创面修复的机制之一。     

DNA binding and topoisomerase I poisoning activities of novel disaccharide indolocarbazoles

The antibiotics AT2433-A1 and AT2433-B1 are two indolocarbazole diglycosides related to the antitumor drug rebeccamycin known to stabilize topoisomerase I-DNA complexes. This structural analogy prompted us to explore the binding of four indolocarbazole diglycosides with DNA and their capacity to interfere with the DNA cleavage-reunion reaction catalyzed by topoisomerase I. The molecular basis of the drug interaction with double-stranded DNA and with purified chromatin, with particular emphasis on the role of the carbohydrate moiety, was investigated by means of complementary spectroscopic techniques, including surface plasmon resonance and electric linear dichroism. We compared the DNA binding properties, sequence recognition, and effects on topoisomerase I-mediated DNA relaxation and cleavage of AT2433-A1 bearing a 2,4-dideoxy-4-methylamino-L-xylose residue, its dechlorinated analog AT2433-B1, the diastereoisomer iso-AT2433-B1 with an inverted aminosugar residue, and compounds 5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, 12-beta-D-glucopyranosyl-12,13-dihydro-6-methyl (JDC-108) and 5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7(6H)-dione, 12-(6-O-alpha-D-galacto-pyranosyl-beta-D-glucopyranosyl)-12,13-dihydro-6-methyl (JDC-277) with an uncharged mono- and disaccharide, respectively. The two antibiotics AT2433-A1 and AT2433-B1 proved to be highly cytotoxic to leukemia cells and this may be a consequence of their tight intercalative binding to DNA, preferentially into GC-rich sequences as inferred from DNase I footprinting studies and surface plasmon resonance measurements. Like the diastereoisomer iso-AT2433-B1, they have no inhibitory effect on topoisomerase I, in contrast to the uncharged diglycoside JDC-277, which stimulates DNA cleavage by the enzyme mainly at TG sites, as observed with camptothecin. Cytotoxicity measurements with CEM and CEM/C2 human leukemia cell lines sensitive and resistant to camptothecin, respectively, also suggested that topoisomerase I contributes, at least partially, to the mechanism of action of the neutral diglycoside JDC-277 but not to that of the cationic AT2433 compounds. Together, the results indicate that sequence-selective DNA interaction and topoisomerase I inhibition is controlled to a large extent by the stereochemistry of the diglycoside moiety.     

Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His₆“tail” at the amino terminus, allowing for purification on a Ni-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6m guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.     

Synthesis of biologically active proteins by recombinant DNA technology

DNA from a variety of sources may be inserted into the DNA of bacteria (cloned) by means of bacteriophage or plasmid vectors. DNA to be cloned may originate from the genome of another organism (genomic DNA), it may be copied enzymatically from messenger RNA (complementary DNA), or it may be synthesized by purely chemical means. In general, cloning of genomic DNA is useful for studying gene structure, but not useful for inducing bacteria to synthesize the proteins coded by the DNA. This is because the genes of higher organisms usually consist of stretches of DNA coding for parts of a protein which are separated from one another by other DNA (intervening sequences) which does not code for protein. Bacterial genes are not interrupted by intervening sequences, hence bacteria cannot make protein accurately from genes containing such interruptions. The use of complementary DNA or chemically synthesized DNA circumvents this problem. These sources of DNA have been used successfully to produce an increasing number of pharmacologically useful proteins. The bacterial synthesis of insulin, growth hormone, and endorphin are reviewed as examples of the applications of this technology.     

Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.     

Production of an Antimicrobial Peptide AN5‐1 in Escherichia coli and its Dual Mechanisms Against Bacteria

AN5‐1 (YSKSLPLSVLNP) is an antimicrobial peptide isolated from the fermentation broth of Paenibacillus alvei strain AN5 (J Ind Microb Biotechnol 2013; 40 : 571–9). In this study, we report the application of ubiquitin fusion technology to the expression and purification of AN5‐1. Minimum inhibitory concentration (MIC) and measurement of hemolytic activity (MHC) were measured to confirm the biological activities of the expressed AN5‐1. Bacterial cell membrane permeabilization was investigated to show the interaction between the AN5‐1 and the bacterial cytoplasmic membrane. Furthermore, intracellular activities of the AN5‐1 were determined by genomic DNA interaction assays. The results revealed AN5‐1 damaging bacterial membranes and binding to bacterial genomic DNA to inhibit cellular functions, suggesting that it has multiple intracellular targets in bacteria. The application of ubiquitin fusion technology may be an excellent approach for industrial production to the expression and purification of antimicrobial peptide. Furthermore, AN5‐1 was demonstrated as an antimicrobial peptide with great potentials, as bacterial resistance to AN5‐1 would be not expected, due to the dual mechanisms of AN5‐1 against bacteria.     

Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice

The administration of butylated hydroxytoluene (BHT) to mice results in lung cell damage followed by cellular proliferation which was quantitated by measuring the increase in thymidine incorporation into pulmonary DNA. Administration of SKF 525-A or piperonyl butoxide to mice treated with BHT prevented the increase in thymidine incorporation into pulmonary DNA. This effect was dose dependent, with complete protection from 400 mg/kg BHT achieved with 10 mg/kg SKF 525-A or 400 mg/kg piperonyl butoxide. SKF 525-A and piperonyl butoxide completely prevented the BHT-induced increase in pulmonary DNA synthesis even when given 1–2 hr after BHT and a partial protective effect was evident when they were given 6–12 hr after BHT. Pretreatment of mice with cobaltous chloride diminished the BHT-induced increase in thymidine incorporation into pulmonary DNA. Following the in vivo administration of [¹⁴C]BHT, radioactivity was covalently bound to lung, liver, and kidney macromolecules of both mice, which exhibited BHT-induced lung damage, and rats, which did not. The greatest amount of radioactivity was bound to lung tissue from mice. This binding was prevented by the administration of SKF 525-A and was a linear function of the BHT dose within a range of 50–600 mg/kg. Binding to other tissues from the mouse and all tissues examined in the rat was minimal and unaffected by SKF 525-A. These data suggest that a reactive metabolite of BHT rather than the parent compound produces lung damage in mice.     

DNA damage repair and response proteins as targets for cancer therapy

The cellular response to DNA damage is critical for determining whether carcinogenesis, cell death or other deleterious biological effects will ensue. Numerous cellular enzymatic mechanisms can directly repair damaged DNA, or allow tolerance of DNA lesions, and thus reduce potential harmful effects. These processes include base excision repair, nucleotide excision repair, nonhomologous end joining, homologous recombinational repair and mismatch repair, as well as translesion synthesis. Furthermore, DNA damage-inducible cell cycle checkpoint systems transiently delay cell cycle progression. Presumably, this allows extra time for repair before entry of cells into critical phases of the cell cycle, an event that could be lethal if pursued with damaged DNA. When damage is excessive apoptotic cellular suicide mechanisms can be induced. Many of the survival-promoting pathways maintain genomic integrity even in the absence of exogenous agents, thus likely processing spontaneous damage caused by the byproducts of normal cellular metabolism. DNA damage can initiate cancer, and radiological as well as chemical agents used to treat cancer patients often cause DNA damage. Many genes are involved in each of the DNA damage processing mechanisms, and the encoded proteins could ultimately serve as targets for therapy, with the goal of neutralizing their ability to repair damage in cancer cells. Therefore, modulation of DNA damage responses coupled with more conventional radiotherapy and chemotherapy approaches could sensitize cancer cells to treatment. Alteration of DNA damage response genes and proteins should thus be considered an important though as of yet not fully exploited avenue to enhance cancer therapy.     

Retinoic acid regulates cell cycle genes and accelerates normal mouse liver regeneration

All-trans retinoic acid (RA) is a potent inducer of regeneration. Because the liver is the principal site for storage and bioactivation of vitamin A, the current study examines the effect of RA in mouse hepatocyte proliferation and liver regeneration. Mice that received a single dose of RA (25 μg/g) by oral gavage developed hepatomegaly with increased number of Ki67-positive cells and induced expression of cell cycle genes in the liver. DNA binding data revealed that RA receptors retinoic acid receptor β (RARβ) and retinoid x receptor α (RXRα) bound to cell cycle genes Cdk1, Cdk2, Cyclin B, Cyclin E, and Cdc25a in mice with and without RA treatment. In addition, RA treatment induced novel binding of RARβ/RXRα to Cdk1, Cdk2, Cyclin D, and Cdk6 genes. All RARβ/RXRα binding sites contained AGGTCA-like motifs. RA treatment also promoted liver regeneration after partial hepatectomy (PH). RA signaling was implicated in normal liver regeneration as the mRNA levels of RARβ, Aldh1a2, Crabp1, and Crbp1 were all induced 1.5 days after PH during the active phase of hepatocyte proliferation. RA treatment prior to PH resulted in early up-regulation of RARβ, Aldh1a2, Crabp1, and Crbp1, which was accompanied by an early induction of cell cycle genes. Western blotting for RARβ, c-myc, Cyclin D, E, and A further supported the early induction of retinoid signal and cell proliferation by RA treatment. Taken together, our data suggest that RA may regulate cell cycle progression and accelerates liver regeneration. Such effect is associated with an early induction of RA signaling, which includes increased expression of the receptor, binding proteins, and processing enzyme for retinoids.