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Natalia LL - artystka neoawangardowa
No full textThe paper shows Natalia Lach-Lachowicz (Natalia LL) as a neo avant-garde artist whose works in a specific maximalistic way are very close to the main currents of avant-garde trends: new mediality (photography), minimalism, conceptualism, performance, bodyart, pop-art, and feminist art. The author of the article concentrates mainly on the mutual influences of conceptualism, consumptionism, and feminism in Natalia LL’s works and pays attention to the emancipatory potential of her works of the seventies and the eighties
Evaluation of production performance and antimicrobial function of the LL-37 peptide as fusion protein and as chemically synthesized and biologically generated peptide
No full text中文摘要
本實驗在評估化學合成的LL-37抗菌胜 (peptide) 和生化合成的LL-37融合蛋白的抗菌功能和生產效益。當人類上皮細胞受到微生物侵入引起局部性發炎時，會產生各種成份的胜，以消滅外來入侵的細菌。其中之一的基因是以豬的PR-39之cDNA作為探針，自人類骨髓cDNA基因庫分離到，此基因的蛋白產物由約170個氨基酸組成，它可被切割成二個片段，N端為cathelin propart，是一種cysteine protein inhibitor，C端為LL-37 antimicrobial peptide，以經過純化的LL-37，進行細菌抑制環分析（inhibition zone assay），顯示其具有抗菌性。它會形成a-helix的結構，且能溶解細菌細胞膜。利用E. coli量產LL-37抗菌胜，將此基因序列插入由125個氨基酸組成的 ketosteroid isomerase (KSI) 下游，及一段His-tag序列的上游。在E. coli表現的KSI/LL-37蛋白質形成的包涵體，自膠體切割回收和分離後，以細菌抑制環分析測試，顯示對革蘭氏陽性及陰性菌均有抗菌活性，但MIC比文獻報導略高。文獻指出，環境因子影響抗菌胜的結構和相異分子間作用的干擾，必須適當調控。接著我們也成功地利用大腸桿菌，表現出一段含三種功能區的LL-37/CBD/RGD融合蛋白質，它可能也受環境影響和融合蛋白的其他部份影響，抗菌效果需進一步研究。Abstract
The aim of this study is to evaluate production performance and biological function of the chemically synthesized LL-37 peptide and the LL-37 as biologically generated fusion protein. Upon infection by microbes, human beings produce antimicrobial peptides as a defense mechanism. One of such genes was isolated from a cDNA library of human myeloid cells by probing with PR-39 cDNA of swine. Protein product of this gene is composed of 170 amino acid residues. It could be subdivided into two fragments. The N-terminal cathelin propart is a cysteine protein inhibitor and the C-terminal LL-37 peptide is an antimicrobial peptide. Inhibition zone assay indicated that purified LL-37 shows antimicrobial activity. Having been demonstrated to form a-helical structure, it could solubilize bacterial membrane. In order to overproduce LL-37 peptide in Escherichia coli, its coding sequence was inserted downstream of a 125 amino acid ketosteroid isomerase (KSI) gene and upstream of a His-tag sequence. The KSI/LL37 overproduced as inclusion bodies was purified from SDS-PAGE gel slice. Inhibition zone assay revealed that it has antimicrobial activity against both gram-positive and gram-negative bacteria, although its MIC appears higher than what has been reported. It has been reported that antimicrobial activity of peptide is influenced by various molecules in the environment and needs to be well controlled. In addition, we also produced in E. coli a tripartite chimeric protein LL-37/CBD/RGD. Its antimicrobial activity, probably also influenced by other components of the chimeric protein, needs to be re-evaluated.[論文目次]
目 次
頁次
中文摘要………………………………………………………………1
英文摘要………………………………………………………………2
壹、 前 言…………………………………………………………..3
貳、 研究動機與目的…………………………….………………...5
參、 文獻探討………………………………………………………6
一、抗菌蛋白(胜)－LL-37的介紹..……………………………6
二、LL-37與抗生素的比較－抗菌機制、抗藥性、優缺點....….9
三、LL-37生產(化學與生化合成方法).………………………….14
四、融合蛋白質的可行性…………………………………………17
五、經濟效益評估…………………….…………………………..19
六、固定化……..……………………………………………….….21
七、研究目的……………………..…………………………….….22
肆、 材料與方法…………………………………………………..…23
一、LL-37化學合成法………………………………………...…..23
二、MIC (minimal inhibitory concentration) 抑制圈的測量…...25
三、LL-37基因片段……………………………………………….26
四、小量DNA質體之抽取………………………………………..26
五、大量DNA質體之抽取………………………………………..27
六、聚合酵素連鎖反應 ( Polymerase chain reaction )……………29
七、DNA的回收與萃取………….…………………………….….30
八、接合作用 (Ligation)………………………………………..….31
九、轉形作用 ( Transformation )………….……………………….32
十、定點突變法 ( Site-Directed Mutagenesis)………………..…...33
十一、重組蛋白的誘導表現…………………………………….....37
十二、不可溶 (KSI) 蛋白質表現系統的建立……………………38
十三、蛋白質膠體 ( SDS-PAGE ) 的電泳分析……………..……39
十四、蛋白質濃度的測定……………..………………………….…40
十五、KSI/LL-37的純化………………………………..……..……40
a. 親合性管柱分析法
b. 電淋洗 (Electro-elution)
十六、溴化氰 (CNBr) 切割生化合成的KSI/LL-37…………….42
十七、LL-37/CBD/RGD蛋白質的純化…………………………….43
十八、可溶 (Heat shock) 蛋白質表現系統的建立………………...44
伍、 結 果 …………………………………………………………...46
一. 化學合成的LL-37抗菌蛋白：
抗菌性分析……………………………………………….. 46
二. 生化合成的KSI/LL-37抗菌蛋白：
1. KSI/LL-37基因的構築……….………………………..46
2. KSI/LL-37的表現及分佈…………….………………..47
3. KSI/LL-37的純化……………………………………...48
a. 親合性色層分析管柱
b. 電淋洗 (Electron-elution)
4. 溴化氰 (cyanogen bromide) 切割含
KSI/LL-37包涵體………………………………………49
5. 胜色層分析管柱純化…..…………….………………50
6. PD-10色層分析管柱純化.………………………………51
7. 最小抑制濃度 (minimal inhibitory concentration)
試驗.………………………………………………….…51
三. 表現 LL-37/CBD/RGD融合蛋白質的基因構築………….51
四. LL-37/CBD/RGD融合蛋白質的誘導與
表現、純化與抗菌試驗………………………………………52
五. pCM7融合蛋白質的誘導表現與純化……………………….53
陸、 討 論 ……………………………………………………………..55
柒、 結 論………………………………………………………………..61
捌、 參考文獻……………………………………………………….…...63
玖、 附錄
培養液與試劑配方……………………………………………..…..70
圖 次………………………………………………………………..73
表 次………………………………………………………………105
圖次
化學合成儀…………………………………………………………….73
LL-37抗菌胜分析…………………………………………………..74
質譜儀鑑定化學合成的LL-37………………………………………..75
化學合成的LL-37的純度…………………………………………….76
化學合成的LL-37抑制大腸桿菌的測試結果…………………...…..77
化學合成的LL-37抑制枯草桿菌與沙門氏菌的測試結果……….…78
AlwNI與Pst I與限制酵素載體進行切割反應………………………79
pET-31b(+) 載體表現系統……..……………………………………..80
LL-37定序圖……………………………………………………….….81
插入pET-31b(+) 系統片段大小的結果………..…………………….82
pHC重組質體構築圖……………………………………………….…83
KSI/LL-37融合蛋白的誘導表現………………………………..…….84
KSI蛋白質的誘導表現………………………………………….…….85
KSI蛋白質的純化結果……………………………………….……….86
KSI/LL-37不可溶蛋白的誘導表現…………………………….……..87
含LL-37包涵體經鎳親合性管柱純化的結果…………..….………...88
電淋洗回收含LL-37包涵體的結果…………………………………..89
溴化氰 (CNBr) 切割LL-37包涵體的結果…………………………..90
化學合成LL-37經過胜色層管柱分析圖…………………………..91
生化合成的LL-37抑制革蘭氏陰性菌 (E. coli) 的測試結果……….92
生化合成的LL-37抑制革蘭氏陰性菌 (Salmonella) 的測試結果…..93
pHC-1載體構築圖……………………………………………………...94
pET-32a(+) 載體表現系統……………………………………..………95
LL-37/CBD/RGD定序圖…………………………………….….……...96
插入pET-32a(+) 系統片段大小的結果………………………….……97
pET表現系統……………………………………………………….…..98 LL-37/CBD/RGD融合蛋白質的誘導與表現……………………….....99
LL-37/CBD/RGD融合蛋白質的誘導與純化…………………….……100
LL-37/CBD/RGD融合蛋白質純化的結果……………………….……101
LL-37/CBD/RGD融合蛋白質抗菌測試……………………………….102
誘導與表現可溶性pCM7融合蛋白質的結果……………………...…103
pCM7融合蛋白質純化的結果…………………………………………104
表次
表一 Primer序列………………………………………………………105
表二 化學合成LL-37的抑菌測試結果………………………………106
表三 市面上胜價格……………………………………..……….….10
Energy flux in isotropic turbulence under large variations of external forcing
No full textWe investigate the response of energy flux in isotropic turbulence to step-function like perturbation in external forcing at large length scales. From both physical experiments and direct numerical simulations, we measured the evolution of the Eulerian velocity structure functions, such as , , before and after the perturbation in forcing. In both cases, we observed the cascade of the energy excess at large scales cascade through scales to the dissipative range, which can be used to study the dynamics of the cascade, and in particular, to estimate the relevant time scales
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No full textStructural and functional analysis of the pro-domain of human cathelicidin, LL-37
No full textCathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of hCLD, LL-37 and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gramnegative bacteria with efficiencies comparable to the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by hCLD intermolecularly, since exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between hCLD and the cysteine protease inhibitor stefin A showed why hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions
An empirical survey: Can green marketing really entice customers to pay more?
No full textThis research integrated the Social Cognition Theory and the Engel Kollat Blackwell customers’ purchasing model (EKB model) to synthetically discuss the three kinds of possible relations comprising “does negatively entice”, “does possibly entice” and “does positively entice” between green-marketing and customers’ purchasing and payment, with consideration given to environmental-protection issues. Based on the measured results, the most contributed contention of this research not only utilized three cross-analytical theories consisting of the social cognition theory (SCT) , the Fuzzy theory (FT) and the EKB model, and the novel F-ANP of the MCDM methodology to evaluate the collected data but it also manifested that Green-marketing does possibly entice customers to pay more (GMPECPM). These measured results have distinctly stunned the fundamental assumption in the traditional green-marketing research field that customers were supposed to be willing to pay more for green products and services because they were supporting green initiatives and helping environmental-protection. Further, major future research directions were also briefly demonstrated in this research as (1) the collection data have to be strengthened to gather more empirical customer feedback, corporate management comments, and professional scholars’ reports; (2) enterprises have to resoundingly establish a green-branding initiative after successfully executing green-marketing strategies.Green Marketing (G-marketing); Multiple Criteria Decision Making (MCDM); Analytical Network Process (F-ANP).
The modulatory effect of TLR2 on LL-37-induced human mast cells activation
No full textThe sole and endogenous anti-microbial peptide LL-37 is a significant effector molecule in the innate host defense system. Apart from its broadly direct anti-microbial activity, the peptide also activates mast cell in respect of allergic diseases and inflammation. On the other hand, mast cell can be activated by Toll-like receptors (TLRs) which are at the center of innate immunity. It was the aim of the study to illustrate the modulatory effect of TLR2 ligands peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4) on LL-37 induced LAD2 cells (a human mast cell line) activation. LL-37 induced LAD2 cells degranulation and the release of IL-8. TLR2 ligands didn't induce LAD2 cells degranulation, but triggered the release of IL-8. Incubation with PGN or Pam3CSK4 significantly suppressed LL-37-induced degranulation through inhibition of calcium mobilization from LAD2 cells. Similarly, the release of IL-8 was inhibited when LAD2 cells were co-stimulated with TLR2 ligands and LL-37. Studies with inhibitors of key enzymes involved in mast cell signaling indicated that the release of IL-8 induced by TLR2 ligands and LL-37 involved the activation of the PI3K, ERK, JNK and calcineurin signaling pathways. In contrast, p38 activation down-regulated the release of IL-8 induced by TLR2 ligands and LL-37. Taken together, these observations suggest that activation of human mast cells by LL-37 could be modified by TLR2 ligands and the function of human mast cells could be switched from allergic reactions to innate immune response. (C) 2016 Elsevier Inc. All rights reserved.National Natural Science Foundation of China [81271755, 81371737]; Guangdong Natural Science Foundation [2014A030313708]; Shenzhen Research Grant [CXZZ20140416144209739, JCYJ20130329110752142, KQCX20120803145850990]SCI(E)[email protected]; [email protected]
The phaeochromycins from streptomyces strain LL-P018: from taxonomy to novelties of biosynthesis
No full textThe phaeochromycins are a newly discovered family of aromatic polyketides produced by Streptomyces strain LL-P018. The novel phaeochromycins A and C are of medical interest as they have inhibitory activity against MK-2, a kinase involved in the inflammatory response. The phaeochromycins are structurally similar to actinomycete secondary shunt metabolites derived from the actinorhodin and enterocin biosynthetic pathways. Because these phaeochromycins show promise as potential anti-inflammatory therapeutic agents, further knowledge of their biosynthesis and the taxonomy of the producing organism was sought. Strain LL-P018 was identified taxonomically using a combination of traditional and molecular techniques. Analyses of morphology, physiology, 16S ribosomal RNA (16SrRNA) sequence, and ribosomal polymerase β- subunit (rpoB) gene sequence were used to identify LL-P018 as a strain of Streptomyces phaeochromogenes. New methods for strain comparison by combining genetic fingerprints and metabolite profiles in a single comparison were developed to assess strain variation between strain LL-P018 and closely related organisms. This approach also clarified relationships between multiple "types" and other published strains of S. phaeochromogenes and Streptomyces ederensis. Alnumycin, an additional aromatic polyketide which has structural similarities to the phaeochromycins, was produced by strain LL-P018 in liquid culture media. Genomic analysis of strain LL-P018 revealed the presence of a type II polyketide synthase gene cluster. Gene disruption experiments determined this pathway to be responsible for both phaeochromycin and alnumycin biosynthesis, suggesting that the phaeochromycins may be intermediates or shunt products in alnumycin biosynthesis.
This thesis describes a novel taxonomic approach to classification which integrates data from prior genetic and metabolic assessments into a single combined comparison. The S. phaeochromogenes type strain is defined, and taxonomic status of the species S. ederensis is clarified. New insights of phaeochromycin biosynthesis are revealed by cultural and genetic studies of Streptomyces strain LL-P018.Ph. D.Includes bibliographical references
LL-37-induced human mast cell activation through G protein-coupled receptor MrgX2
No full textHuman LL-37 is an important class of cationic antimicrobial peptide (CAP) that is known to stimulate mast cell activation. While many studies have been conducted on LL-37, to date little is known about the functional receptors for LL-37-induced human mast cell activation, in particular in terms of the release of de novo synthesized mediators. Thus, the aim of the present study is to identify the functional receptors for LL-37-induced human mast cell activation in terms of the degranulation and release of de novo synthesized mediators and investigate the downstream signalling pathways involved in mast cell activation. Overall, our study importantly demonstrates that LL-37-induced human mast cell degranulation and release of de novo synthesized mediators function primarily through the activation of MrgX2. We furthermore show that LL-37-induced human mast cell line LAD2 cells are involved in the degranulation and release of IL-8, and that FPRL1 and P2X7 have only a partial effect on IL-8 release, and no effect on mast cell degranulation triggered by LL-37. Instead, we find that silencing the expression of MrgX2 in human mast cell significantly inhibits the LL-37-induced degranulation and release of IL-8. Overall, this effect is associated with the activation of the Gi protein, PLC/PKC/Calcium/NFAT, PI3K/Akt and MAPKs signalling pathways.National Natural Science Foundation of China [81401299]; Natural Science Foundation of Guangdong Province [2014A030313711]; Characteristic innovation project of Guangdong Province Ordinary University [2015KTSCX120]; Shenzhen Peacock Plan [827-000209]SCI(E)ARTICLE6-124
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