Dissertations / Theses on the topic 'Antifungal antibiotic'

The antifungal polyene macrolide nystatin is produced by Streptomyces noursei ATCC 11455. The nystatin biosynthesis gene cluster of Streptomyces noursei has been cloned and sequenced, and a biosynthesis route has been predicted. In the present work, investigation of genes presumably involved in post-PKS modifications of nystatin is described. The aim of this work was to better understand the nystatin biosynthesis and to further use this information for generation of novel nystatin analogues. Two PKS-modifications of the nystatin molecule were targeted in this study: glycosylation with mycosamine at C-19 and oxidation of the exocyclic methyl group at C-16.

Two genes putatively involved in mycosamine biosynthesis (NysDIII and NysDII) and one in attachment of mycosamine to the nystatin aglycone (nysDI) have been identified in the nystatin gene cluster. Their functions have been suggested, respectively, as a putative mannose dehydratase, aminotransferase and a glycosyltransferase. The deoxysugar mycosamine is proposed to have an important function for the activity of nystatin. To better understand the biosynthesis and importance of mycosamine and to perform modifications of nystatin via this post-PKS modifying step, the mycosamine biosynthesis was studied. The NysDIII protein was overexpressed in Escherichia coli and purified, and its in vitro mannose 4,6-dehydratase activity was confirmed. To study the function of nysDII and nysDI, the genes were individually deleted from the S. noursei chromosome. Both mutants were shown to produce a mixture of nystatinolide and 10-deoxynystatinolide, albeit at considerably different levels. Complementation experiments unequivocally confirmed the involvement of these two in mycosamine biosynthesis and attachement. Both antifungal and hemolytic activity of the purified nystatinolides were tested, and were found to be strongly reduced compared to nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

A gene for putative P450 monooxyganse NysN has been identified in the nystatin biosynthesis gene cluster. The function of NysN has been predicted to be oxidation of an exocyclic C16 methyl group on the nystatin molecule in order to afford a C16 carboxyl. The latter group has been implicated in selective toxicity of other polyene macrolides, and thus is considered an important target for manipulation. The nysNgene was inactivated in S. noursei by both in-frame deletion and site-specific mutagenesis, and the resulting mutants were shown to produce 16-decarboxy-16-methylnystatin, supporting the suggested biological role of NysN as C16 methyl oxidase. The recombinant NysN protein was also expressed in Escherichia coli, but its C16-methyl oxidase activity in vitro could not be demonstrated. 16-decarboxy-16-methylnystatin was purified from the nysN mutant, and its antifungal activity was identical with nystatin whereby the toxicit was reduced compared to nystatin.

In the work of developing new methods for obtaining nystatin analogues, bioconversion of nystatinolide was performed as a means to modify nystatin aglycone. For this purpose a sub-library of 35 different Streptomyces strains isolated from the Trondheims fjord was selected. One strain was shown to be able to add a water molecule (presumed epoxidation) and another strain was able to chlorinate the nystatinolides. An attempt on alternative glycosylation of nystatinolide was performed by using glycosyltransferase hybrids and deoxysugar biosynthesis gene cassettes. However, these experiments did not afford novel nystatin analogues, suggesting strong preference of the NysDI glycosyltyransferase for its natural sugar substrate GDP-mycosamine.

The polyene macrolide antibiotic natamycin (Antibiotic A-5283) is commonly used to retard the growth of surface molds on various cheese varieties. Natamycin is commonly applied to the surface of cheese by dipping or spraying, using an aqueous dispersion containing 200 to 300 ppm of the additive. The large molecular weight of natamycin, 666 g/mol, and conjugated double bond structure causes it to be extremely insoluble in water and most food grade solvents. The inability to apply natamycin in true solution creates void non-treated areas on the food surface. These non-treated areas promote the growth of fungal organisms. A water soluble N-alkyl semisynthetic derivative of natamycin was synthesized by the Michael addition reaction of the parent with a N-substituted malemide. A comparative study investigating the effectiveness of the semisynthetic derivative of natamycin and the parent antibiotic in suppressing mold growth on one month aged shredded Cheddar cheese modified atmosphere packaged (MAP) was performed. A 20 ppm natamycin treatment effectively suppressed visible mold growth (<104 CFU/g) in MAP samples for up to 30 days after opening. The 20 ppm semisynthetic derivative performed similarly to the 10 ppm natamycin treatment in retarding mold growth. Visible mold growth did not occur for these treatments in MAP samples until 20 days after opening. Analysis of storage conditions revealed that an outgrowth of mold in shredded cheese occurred in MAP packages stored longer than 15 days. This bloom in mold growth was attributed to the degradation of natamycin and the semisynthetic derivative throughout storage. The stability and degradation of natamycin and the derivative were monitored throughout the study. Antibiotic concentration on the cheese surface was quantified by molecular absorption spectrometry. Results from this study showed, heavily contaminated samples caused the rate and loss of natamycin and the derivative to increase. Antibiotic concentration decreased at a similar rate in MAP and open package conditions. Natamycin and derivative were found to have similar degradation properties.
Master of Science

Thesis (PhD) -- University of Western Sydney, 2007.
A thesis submitted to the University of Western Sydney, College of Health and Science, School of Natural Sciences, as a requirement for the degree of Doctor of Philosophy. Includes bibliography.

N-Thiolated β-lactams (1) represent a promising new group of compounds with potent inhibition effects on bacteria, like Bacillus anthracis and methicillin resistant Staphylococcus aureus, and onco-systems, like breast cancer and leukemia. Originally developed as part of a synthetic pathway to bicyclic lactams, N-thiolated β-lactams have been shown in this laboratory to possess intriguing biological activities. The antibacterial activities of this new class of agents rely on novel structural features unlike those of any existing family of β-lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active β-lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes but instead pass through these membranes. The biological target of these compounds has been investigated. The most active members of this β-lactam class appear to be those bearing a small branched alkyl chain on the sulfur atom. The effects of stereochemistry, branching and chain length of the sulfur group on bioactivities were studied. This dissertation is divided into six chapters. A review of organosulfur anti-infectives is discussed in Chapter 1. The types of existing antibiotics and their modes of action will be discussed in Chapter 2. The synthesis of these novel agents is discussed in Chapter 3. A structure-activity relationship of these lactam analogues is discussed in Chapter 4. And Chapters 5 and 6 demonstrate a novel mode of action and biological target for these drugs using techniques which include target identification, metabolic effects, and reactivity kinetics.

Thesis (M Med (Otorhinolaryngology))-- University of Limpopo, 2011.
Aims: The purpose of this study was to assess if the use of post-operative antibiotics have any beneficial effects in reducing morbidity following elective tonsillectomy in children with age range of 1-13 years. . Objectives: To assess the degree of post-tonsillectomy pain, determine the incidence of secondary haemorrhage, establish the time period to the resumption of a normal diet, document adverse effects of the use of antibiotics (e.g. skin rash, anaphylaxis, diarrhoea and vomiting), determine the bacteriology in tonsil removed and make recommendations on post-tonsillectomy treatment protocol at DGMH. Methods: This prospective study was conducted at DGMH on 81 children with an age range of l-13years (mean 5.7years). At total of 40 children received paracetamol l5mg/kg/day (Group A) in three divided doses for seven days, and 41 received amoxicillin 40 mg/kg/day and paracetamol (Group B) for the same duration. The post operative morbidity and bacteriology of the two treatment groups were compared. Primary outcomes measured included the incidence and severity of pain, use of analgesia, resumption of normal diet, incidence ofheamorrhage, fever, vomiting and adverse reactions.

These studies have characterized the peptide transport systems of Candida albicans, with a view to the rational design of peptide antifungal agents exploiting the 'smugglin' concept. In initial studies, a series of polyoxin complexes (peptide-nucleoside antibiotics) and individual components, were isolated from a batch of agricultural fungicide (Polyoxin Z). Isolated fractions were toxic to a particulate chitin synthetase preparation from Candida albicans. Different strains of Candida albicans exhibited varied sensitivities to a series of peptide analogues. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross-resistant to the other analogues and to m- fluorophenylalanylalanylalanine a but retained sensitivity to m- fluorophenylalanylalanylalanine. This mutant showed defective dipeptide transport but normal oligopeptide transport, and was unable to utilize Ala-Ala as a sole nitrogen source. Thus, Candida albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts. Peptide transport was shown to be stimulated by the presence of peptides (peptone) in the growth medium. On transferring cells from minimal to peptone medium, this stimulatory effect was shown to be rapid, independent of protein synthesis and to override ammonia regulation of peptide transport. The reduction of transport activity on transferring cells from peptone to minimal medium was also rapid. It was speculated that regulation of peptide transport is achieved by a rapid, reversible activation of preformed transport components, or a mechanism of exocytotic insertion and endocytic retrieval of preformed transporters. The effect of protein-modification reagents on transport activity was also examined. Dipeptide transport was specifically inhibited by N-ethyl-5-phenylisoxazolium-3'-sulphonate (Woodwards Reagent K), offerring potential for the specific labelling of the component(s) of this system. Peptide transport was shown not to be sensitive to osmotic shock though a series of uncharacterized polypeptides was released by the shock treatment.

Le cycloheximide ou actidione, antibiotique antifongique polyvalent, est non bactéricide. La CMI sur une levure sensible, saccharomyces uvarum, a été déterminée. Les études réalisées sur la structure pariétale de rhodotorula glutinis CBS 3044 incubée en présence ou en absence du cycloheximide ont révélé l'existence du fucose, du galactose, du glucose et du mannose. Deux types de peptidohétéromannannes ont été constatés dans les parois RH. Glutinis CBS 3044 : le type alpha, de MMA élevée, pauvre en aminoacides, mais riche en galactose, le type beta, de MMA plus faible, riche en aminoacides mais pauvre en galactose. Seule la synthèse des peptidohétéromannannes extraits par la méthode de peat n'ont fait que confirmer l'existence des composés alpha et beta

Synthesizing bioactive small molecules by structural modification of 1,4-naphthoquinone was the primary goal of this research. Several bioactive compounds with anticancer, antifungal, and antibacterial activities were synthesized. All the synthetic protocols were optimized in such ways that do not require cumbersome purification. First, a new protocol for the synthesis of NQM111 was developed. NQM111 is a highly potent anticancer agent developed in our laboratory, but the old protocol does not provide enough quantity for in vivo study. Therefore, a new safe and improved method was developed which provides enough quantity for in vivo study. The second project involves the synthesis of 1,4-naphthoquinone conjugated with an aromatic group. These compounds are a highly potent anticancer agent with ~8-fold selectivity towards cancer cell lines than the non-cancer cell line. A mode of action study of this compound was identified, and it was observed that these compounds generate reactive oxygen species,which triggers apoptosis. The final project involves the synthesis of 1,4-naphthoquinone based antifungal, and antibacterial compounds. These compounds are multi-cationic in nature with a hydrophobic tail. Six different analogs with varying hydrophobic tails were synthesized and tested for their antibacterial and antifungal activity. These compounds showed excellent activity against wide range of fungi including resistant strains.

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

Thesis (MTech Biomedical Technology)) ---Cape Peninsula University of Technology, 2008
Resistance to antibiotics has been acknowledged as a major global public health problem. The use of peptides to provide alternatives to combat multi drug resistant organisms is of current relevance to overcome antibiotic resistance. The high deversity of amphibian skin peptides render these animals a potential source for the discovery of novel drugs.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The global rise in microbial resistance, ranging from the agricultural industry to the medical sector, has created the urgent need for novel or supplementary antibiotics. Antimicrobial peptides or “nature’s antibiotics” may be the answer to this major problem. In this study a group of antimicrobial peptides, cyclic decapeptides named tyrocidines, produced by the soil bacterium Bacillus aneurinolyticus, was investigated for their antifungal activity, possible mode of antifungal action and potential applications. The study illustrated that the tyrocidines have significant antifungal activity against a range of phytopathogens, including Fusarium solani and Botrytis cinerea, as well as the human pathogen Candida albicans. The activity of the tyrocidines is influenced by the identity of both the target organism and the media environment. Further evidence was obtained in support of the hypothesis that the tyrocidines are extremely sensitive to their environmental conditions and that they tend to self-assemble to form oligomers. The assessment of a small tyrocidine library and analogues, comprised of eight peptides, revealed no overt structure-activity relationships against fungal pathogens, except for the importance of a tyrosine residue. This indicated an important role for the conserved sequence of the tyrocidines, NQYVOLfP, together with the tendency of the tyrocidines to oligomerise into higher-order active structures in their antifungal activity. The tyrocidines were found to be membrane active toward the fungal pathogens. However, supporting evidence was also obtained for additional mode(s) of antifungal action for the tyrocidines which inter alia induces morphological abnormalities in filamentous fungal target cells. Furthermore, the results also indicated that the membrane activity of the tyrocidines may be influenced by additional factors to that of the composition of the target cell membrane, for instance components of the fungal cell wall. This investigation also indicated the significant potential of the tyrocidines to be developed for the commercial sector. The potent activity of the tyrocidines against agronomically important phytopathogens (significantly higher than the commercial fungicide bifonazole) together with their relative salt stability bodes well for their development as bio-fungicides for the agricultural sector. The tyrocidines also exhibited an overt sinergistic effect on the in vitro candidacidal activity of two key antifungal drugs, caspofungin and amphotericin B. Furthermore, tyrocidine A and caspofungin exhibited synergistic activity in vivo which had a significant positive effect on the survival of C. albicans infected Caenorhabditis elegans. Latter results highlighted their potential to serve as candidates for combinatorial treatment in the medical industry.
AFRIKAANSE OPSOMMING: Die globale verskynsel van mikrobiese weerstand, wat strek vanaf die landbou sektor tot in die mediese bedryf, het ’n dringende behoefte vir die ontwikkeling van nuwe antmikrobiese middels geskep. Antimikrobiese peptiede of “die natuur se antibiotika”, kan moontlik die antwoord op hierdie ernstige problem wees. Tydens hierdie studie is ‘n groep sikliese antimikrobiese peptiede, naamlik die tirosidiene wat deur die grondbakterium Bacillus aneurinolyticus geproduseer word, vir hulle antifungiese aktiwiteit, hulle moontlike meganisme(s) van antifungiese werking en hulle potensiёle aanwendings bestudeer. Hierdie studie het getoon dat die tirosidiene uitsonderlike antifungiese aktiwiteit teen ‘n reeks fitopatogene, insluitend Fusarium solani en Botrytis cinerea, asook teen die mens patogeen Candida albicans het. Die aktiwiteit van die tirosidiene is deur beide die identiteit van die teikenorganisme sowel as die mediumomgewing beїnvloed. Daar is ook verdere bewyse verkry wat die hipotese dat tirosidiene uiters sensitief is tot hulle omgewing en dat hulle neig om te oligomeriseer, ondersteun. Die studie van die klein tirosidien-biblioteek, saamgestel uit agt tirosidiene en analoё, het geen ooglopende struktuur-aktiwiteit verwantskappe opgelewer nie, behalwe vir die oёnskynlike invloed van die tirosien-residu. Laasgenoemde het die belangrikheid van die gekonserveerde aminosuurvolgorde van die tirosidiene, NQYVOLfP, asook die neiging van tirosidiene om hoё-orde aktiewe strukture te vorm deur self-verpakking, beklemtoon. Tydens die studie is daar gevind dat die tirosidiene membraan-aktiewiteit toon teenoor fungiese patogene. Daar is egter ook goeie bewyse vir alternatiewe meganisme(s) van antifungiese werking, wat ondermeer tot morfologiese abnormaliteite in filamentagtige fungi-teikenselle lei, vir die tirosidiene verkry. Die resultate het verder ook daarop gewys dat die membraan-aktiwiteit van die tirosidiene ook deur ander faktore, soos deur komponente van die fungiese selwand, en nie net deur die samestelling van die fungiese membraan beїnvloed word nie. Hierdie ondersoek het ook die aansienlike potensiaal van die tirosidiene vir kommersiёle ontwikkeling en gebruik uitgelig. Die merkwaardige aktiwiteit van die tirosidiene teen fitopatogene van agronomiese belang (wat selfs beter as diè van die kommersiёle swamdoder bifonazole was) tesame met die relatiewe sout stabiliteit van die tirosidiene, is belowende tekens om die tirosidiene as bio-swamdoders vir die landbou sektor te ontwikkel. Die tirosidiene het ook ‘n uitgesproke sinergistiese effek op die in vitro candidasidiese aktiwiteit van twee sleutel antifungiese middels, caspofungin en amphotericin B, getoon. Verder is daar in vivo sinergistiese aktiwiteit gewys deur die kombinasie van tirosidien A en caspofungin wat ’n beduidende positiewe effek op die oorlewing van C. albicans geïnfekteerde Caenorhabditis elegans gehad het. Laasgenoemde dui op die potensiaal van die tirosidiene om in die mediese bedryf as kandidate vir kombinasie-behandeling te dien.

碩士
國立中正大學
化學工程研究所
84
The technology of microencapsulation has developed for several decades. Many industries have been concerned with microencapsulated products. Development of the polymers for microencapsulation classically emphasized the technology to make materials durable and weatherable. During the 1970's a need arose of biodegradable materials for drug, agricultural and environment protection requirements. But the existing biodegradable materials are too expensive for the agricultural purpose. Furthermore, the chemical pesticides have brought many serious damages to the environment, so the most important thing to the agriculture is the introduction of the microbial pesticides. In this research, we developed the techniques of microencapsulating biologically antifungal antibiotic using the biodegradable polymers as the coating materials by a simple coacervation-phase separation procedure. The coating materials are gluten and casein. The core materials are either the cell powder containing the biologically antifungal antibiotic, pyrrolnitrin, or the Microken powders adsorbing pyrrolnitrin. The irregular microcapsules prepared in this research have a particle size range between 5-80 , and a smooth surface after hardening. In the degradation experiments of coating materials we found that in twenty days the degradation of gluten particles without hardening was 29.6﹪and with hardening 12.9﹪,whereas the degradation of casein particles without hardening was 70.4﹪ and with hardening 38.2﹪. When the cell powders were capsulated with gluten, six or more times delay in the release of pyrrolnitrin was achieved and with casein had a five or more times delay was observed. When the adsorbed Microken powders were capsulated with gluten and casein and then hardened, the delay of releasing pyrrolnitrin were respectively, 4.7 and 8 times in comparing with releasing non-encapsulated pyrrolnitrin. Besides, the release rates of microcapsulates fit well the first-order release model and the Higuchi model.

碩士
大同工學院
化學工程研究所
87
A antifungal antibiotic producing bacterium strain AF-714, isolated from out door soil was identified as Gram negative. AF-714 strain produced the antifungal antibiotic extracellularly when incubated in liquid medium with 3% sucrose and 0.5% soybean for 20 hours. The antifungal antibiotic was purified from the crude extract of producing broth by acetone precipitation, Fractogel DMAE-650M column, Charcoal activated filtration, Biogel P-2 column, Charcoal activated column, Sephadex G-15 column and Silica gel 60 PLC. The purified antifungal antibiotic was obtained, with a recovery of 17%, and shows as a white color powder. The antifungal antibiotic showed extreme hydrophilicity and low molecular weight ( about 1000 ). The antifungal activity is no loss in the temperature range of 0℃to 100℃for 30 minutes. The purified antifungal antibiotic was activated even after incubation at 30℃ in organic solvent for 2 hours. It is active against Candida albicans and filamentous fungi such as Athelia rolfsil and Rhizoctonia solani.

碩士
國立臺灣大學
植物病理與微生物學研究所
105
The genus Fusarium comprises many species, including F. oxysporum, F. solani, F. graminearum, and F. verticillioides, and causes severe infections in plants and humans. In clinical settings, Fusarium is the third most frequent mold to cause invasive fungal infections, after Aspergillus and the Mucorales. F. solani and F. oxysporum are the most prevalent Fusarium species to cause clinical disease. However, few effective antifungal drugs are available to treat both human and plant Fusarium infections. The cationic peptide antibiotic polymyxin B (PMB) exhibits antifungal activity against the human fungal pathogens Candida albicans and Cryptococcus neoformans, but its efficacy against Fusarium species is unknown. In this study, we tested the antifungal activity of PMB against 12 Fusarium strains that infect humans and plants (banana, tomato, melon, pea, wheat, and maize). PMB was fungicidal against all 12 Fusarium strains, with minimum fungicidal concentrations of 32 or 64 μg/mL for most strains tested, as evidenced by broth dilution, methylene blue staining, and XTT reduction assays. PMB can reduce the germination rates of conidia, but not chlamydospores, and can cause defects in cell membrane integrity in Fusarium strains. PMB exhibits synergistic activity with posaconazole, and can potentiate the effect of fluconazole, voriconazole, or amphotericin B against Fusarium species. However, PMB does not show synergistic effects with fluconazole against Fusarium species as it does against Candida glabrata and C. neoformans, indicating evolutionary divergence of mechanisms between yeast pathogens and the filamentous fungus Fusarium.

博士
國立陽明大學
生物化學研究所
88
The aims of this study are to screen the novel antifungal antibiotic and glycoprotein intracellular transport inhibitor. It divied into three parts: 1, the screening for the strains of Actinomycetes, 2, the screening for novel antifungal antibiotics, and 3, screening for the glycoprotein intracellular transport inhibitor. In this study, we collected 85 soil samples from Taiwan island and 188 feces samples from Taipei City Zoo. After using 5 soil pretreatment including dry heat treatment, moisture heat treatment, SDS-yeast treatment and phenol treatment and 5 isolation mediums including M3, AY, GA, HV and AI, we isolated 1847 strains of actinmycetes from those soil and feces samples. Those actinomycetes were fed into our screening systems for searching antifungal antibiotics and the glycoprotein transport inhibitors. We use antifungal susceptibility test of Candida albican as the frist screening system for antifungal antibiotics. These 1847 actinomycetes were screened and selected 104 strains for antifungal activity against Candida albican. Then, the plant pathogenic fungi, Pythium splendens、Rhizoctonia solani and Phytophthora capsici, were used ass the test organisms for antifungal activity screening. By antifungal susceptibility test, these 104 actinomycetes were screened for antifungal activity against plant pathogenic fungi. A strain, 84A22, identified as Streptomyces alboviridis, against Pythium splendens and Phytophthora capsici was selected to purify its antifungal compound. The pure compound was obtained from fermentation broth by extraction with ethyl acetate, silica gel column chromatographies, and HPLC. The molecular weight of this compound is 1100 by mass spectrometry. Its structure is determined by NMR spectrometry and named as alboviridimycin.. Using the screening system mentioned below we try to identify the unique and specific inhibitors for glycoprotein transport from Actinomycetes. This screening system was constructed by using baby hamster kidney cell (BHK-21 cell) and New Castle Disease Virus (NDV) for screening the glycoprotein''s inhibition and presentation produced by virus. Syntium form-ation and hemaglutination are the two phenomena caused by the glycoproteins produced by NDV after infection. The F protein and HN protein produced by virus are the causes of syntium formation and why hemaglutination take place. We use microscope to observe the syntium formation to determine the F protein''s transportation and use the hemaglutination test to assay the presentation of HN protein in the cytosal. We select the substance that can inhibit the syntium formation caused by the transportation of F proteins block but cannot stop the HN protein synthesis. We used this screening system to select inhibitors from Actinomycetes and selected several candidates for further identification and characterization. And now, we choose the strain 48P1I2 which was identified as Streptomyces gougerotii as the production strain. After purification process, we get the purified compound and try to estimate its structure. At the same time, we also study the molecular mechanism of the inhibition of the glycoprotein intracellular transport inhibitor ABSTRACT 8 縮寫 9 第一章、緒論 9 第一節、放線菌(Actinomyces) 10 一、放線菌(Actinomycetes)簡介 10 二、放線菌與生理活性物質之篩選 11 三、放線菌之工業用途 13 第二節、抗真菌劑 13 一、抗真菌劑簡介 13 二、抗真菌劑之作用機制 14 1、 作用在固醇類（sterol）生合成及功能上的抗真菌劑 14 2、作用在細胞核功能上的抗真菌劑 15 3、抑制真菌細胞壁生合成的抗真菌劑 15 4、作用在能量代謝的抗真菌劑 15 三、醫藥用抗真菌劑 15 1、 Polyenes系列抗真菌劑 16 2、Azoles系列抗真菌劑 16 3、Glucan 合成抑制劑 16 4、Chitin 合成抑制劑 16 5、Nucleoside 類似物 17 四、農業用抗真菌劑(William, 1986) 18 五、peptide類抗真菌劑(Robert and Daniel, 1999) 18 第三節、醣蛋白質細胞內輸送阻害物質 19 一、醣蛋白質細胞內輸送途徑 19 二、醣蛋白質細胞內輸送程序之解明 20 1、利用Saccharomyces cerevesia 蛋白輸送分泌變異株之研究法 20 2、利用動物細胞醣蛋白分泌阻害變異株之研究法[Tufaro, 1987 #172] 20 3、醣蛋白質細胞內輸送機制之in vitro 研究法 21 4、應用選擇性醣蛋白質細胞內輸送阻害劑之研究法 21 三、醣蛋白質細胞內輸送阻害劑及其應用(Takatsuki and Tamura, 1985) 21 四、醣蛋白質細胞內輸送阻害劑之應用 23 第四節、研究策略 24 第二章、材料與方法 26 第一節、材料 26 一、培養基及培養液 26 1、放線菌篩選培養基(Orchard and Goodfellow, 1974; Wakasman, 1961) 26 2、抗真菌劑篩選培養基 27 3、放線菌鑑定用培養基 29 4、醣蛋白胞內輸送抑制物發酵培養基 30 5、動物細胞培養基 31 二、菌種、細胞株、病毒及抗體： 31 1、抗菌活性檢定用 31 2、醣蛋白胞內輸送抑制物檢定 32 三、試藥及耗材： 33 1、化學藥品： 33 2、培養基及佐劑： 33 3、有機溶劑： 33 4、Paper disc： 33 四、層析管柱： 33 第二節、方法 35 一、篩選種源庫之建立 35 1、菌種分離 35 2、菌種培養與物質萃取 36 二、放線菌菌種鑑定 37 1、形態觀察： 37 2、細胞壁組成分析： 37 三、抗真菌劑之篩選與純化 38 1、對峙培養法： 38 2、paper disc agar diffusion assay： 38 3、醱酵培養 39 4、活性物質之萃取與純化 40 5、結構鑑定： 42 6、感受性測試： 43 7、細胞毒性測試(Tada et al., 1986; Veronique et al., 1992)： 44 四、醣蛋白質細胞內輸送抑制物質之篩選 45 1、抑制物質之篩選方法(Muroi et al., 1996; Seog et al., 1994) 45 2、醱酵培養 46 3、醣蛋白胞內輸送抑制物質純化 46 4、醣蛋白胞內輸送抑制物質鑑定 49 5、醣蛋白胞內輸送抑制物質機構 49 第三章、結果 52 第一節、放線菌菌株篩選 52 一、菌種分離 52 1、樣本採集： 52 2、樣本前處理(Steele, 1991)︰ 53 3、菌株分離培養基： 54 二、糞便樣本之放線菌菌株分離統計： 54 三、放線菌菌屬鑑定 55 1、形態觀察： 56 2、細胞壁組成分析： 56 第二節、活性物質之篩選 57 一、第一次對峙培養篩選 57 二、活性菌種鑑定 59 三、以植物病源菌為檢定菌之二次篩選 59 1、對峙培養法： 60 2、paper disc agar diffusion assay(Tamura et al., 1968)： 61 3、菌種鑑定： 61 第三節、Streptomyces alboviridis 84A22之抗真菌物質 62 一、醱酵培養： 62 二、84A22抗真菌物質之萃取與純化 63 1、第一次Silica gel 管柱層析： 63 2、第二次Silica gel 管柱層析： 63 3、製備級silica gel HPLC： 63 4、 分析級silica gel HPLC： 63 5、C18 HPLC： 64 6、phenyl HPLC： 64 三、結構鑑定 64 1、UV spectrum 64 2、mass spectrum 64 3、1H NMR spectrum 64 4、13C NMR spectrum 64 5、2D-NMR 65 6、FT-IR spectrum 65 7、amino acid composition 65 四、抗真菌劑抗菌性測試 66 1、細菌對alboviridimycin之感受性測試 66 2、酵母菌對alboviridimycin之感受性測試 66 3、alboviridimycin對黴菌之感受性測試 66 4、alboviridimycin對細胞之細胞毒性測試 67 第四節、醣蛋白細胞內輸送抑制物質之篩選 67 一、醣蛋白細胞內輸送抑制物質之篩選 67 二、48P1I2之菌種鑑定 68 1、細胞壁成分分析 68 2、形態特徵 68 3、醣類的利用及生理特性測試 68 4、菌絲與孢子之電子顯微鏡觀察 68 三、醣蛋白質細胞內輸送抑制物質GPT48之發酵培養 69 四、GPT48之萃取與純化： 69 1、Silica gel 管柱層析 I 69 2、Silica gel Silica gel 管柱層析 II 70 3、製備級silica gel HPLC： 70 4、分析級silica gel HPLC： 70 5、C18 HPLC： 70 6、phenyl HPLC： 70 五、醣蛋白胞內輸送抑制物質GPT48結構鑑定 71 1、mass spectrum 71 2、1H NMR spectrum 71 六、醣蛋白胞內輸送抑制劑GPT48抑制機制 71 1、GPT48處理後VSV-G 蛋白於細胞內蓄積部位之分析： 71 第四章、討論 74 第一節、放線菌菌種分離 74 第二節、抗真菌物質之篩選 76 一、抗真菌劑生產菌株之篩選 76 1、篩選方法 76 2、形態異常誘發 77 二、Streptomyces alboviridis之抗真菌劑alboviridimycin 78 1、Alboviridimycin之純化與結構鑑定 78 2、alboviridimycin之抗菌特性 79 3、alboviridimycin對細胞之細胞毒性測試 80 4、後續之研究 80 第三節、醣蛋白胞內輸送抑制物質之篩選 81 一、醣蛋白胞內輸送抑制物質之篩選 81 二、Streptomyces gougerotii之醣蛋白胞內輸送抑制物質GPT48 82 1、GPT48之純化與結構鑑定 82 2、GPT48之特性 83 3、後續之研究 86 第五章、參考文獻 87 第六章、附錄 100

Encapsulation is a novel technique that involves the entrapment of materials such as cells, enzymes or chemicals within a semi-permeable matrix and is being explored as a drug delivery system. This project investigated the encapsulation of Streptomyces nodosus in alginate to assess whether this organism can produce the antifungal drug amphotericin B from within the matrix. New methods were developed to immobilise S. nodosus mycelia and spores in alginate capsules, assess bacterial viability and detect ng mL–1 quantities of amphotericin B in culture fluids. When capsules were cultured and cell proliferation was encouraged, organisms formed protrusions on the surface of the capsules. Differentiated branched hyphae that never progressed to sporogenic hyphae were observed on the surface of these structures. Viability was maintained for up to 30 days and low levels of amphotericin B were produced. The emergence of a co-existing free-dwelling population was also observed. Culturing immobilised organisms using conditioned media from an amphotericin deficient S. nodosus strain, augmented the development of the free-dwelling population resulting in the detection of amphotericin B in the culture fluid and full differentiation to sporogenic hyphae. This is the first report of sporulation of S. nodosus in liquid environments and demonstrates that immobilised S. nodosus can produce antibiotics. The sporulation of free-dwelling organisms was also induced using conditioned media and manipulation of quorum size, indicating a solid surface is not required for sporulation. Conditioned media from other Streptomyces spp. induced variable responses including sporulation, pigment formation and antibiotic production, possibly demonstrating communication between species and/or alteration in nutritional status. This new model for the life cycle of S. nodosus will permit the study of developmental pathways, antibiotic production, microbial community structure and inter-species and intra-species signalling.
Doctor of Philosophy (PhD)

碩士
國立陽明大學
生物化學研究所
87
Fungicides are often used to control fungal diseases for plant protection in agriculture. Because of the increasing arise of drug resistant fungi and most of the fungicides are not biodegradable that causes residual toxicity to other organism. The development of new antifungal agents that possess low toxicity, high efficacy, and high selectivity is necessary. In this study, methods for the isolation of bacterial strains from animal feces is evaluated. From a collection of 10 different animal feces, a total of 459 Actonimycetes strains were isolated. We found that pretreatment of animal feces with SDS-yeast extract and use of M3, A1, or HV media gave better recovery of Actinomycetes strains. In addition, using phytopathogenic fungi Pythium splendens, Rhizocotonia solani, and Phytophthora capsici as the tested strains for antifungal agent-screening model, the antifungal activity of 48 laboratory antifungal strains were analyzed. These 48 strains were previously collected for their inhibitory activity in Candida albican growth. The culture medium prepared from strain 84A22, a Streptomyces alboviridis, inhibited the growth of both P. splendens and P. capsici. The active component existing in the culture medium of 84A22 was purified by ethyl acetate extraction, silica gel column chromatography, and HPLC. Mass spectrometric analysis indicated that the molecular weight of this active component is ~1100. Determination of the structure of this active component is currently in progress.

Infectious diseases caused by bacteria, fungi, and plasmodium parasites are a huge global health problem which ultimately leads to millions of deaths annually. The emergence of strains that exhibit resistance to nearly every class of antimicrobial agents, and the inability to keep up with these resistance trends has brought to the fore the need for new therapeutic agents (antibacterial, antifungal, and antimalarial) with novel scaffolds and functionalities capable of targeting microbial resistance. A novel class of compounds featuring an aryl isonitrile moiety has been discovered that exhibits potent inhibitory activity against several clinically relevant strains of methicillin-resistant Staphylococcus aureus (MRSA). Synthesis, structure-activity relationship (SAR) studies, and biological investigations have led to lead molecules that exhibit anti-MRSA inhibitory activity as low as 1 – 2 µM. The most potent compounds have also been shown to have low toxicity against mammalian cells and exhibit in vivo efficacy in MRSA skin and thigh infection mouse models.

The novel aryl isonitriles have also been evaluated for antifungal activity. This study examines the SAR of aryl isonitrile compounds and showed the isonitriles as compounds that exhibit broad spectrum antifungal activity against species of Candida and Cryptococcus. The most potent derivatives are capable of inhibiting growth of these pathogens at concentrations as low as 0.5 µM. Notably, the most active compounds exhibit excellent safety profile and are non-toxic to mammalian cells up to 256 µM.

Beyond the antibacterial and antifungal activities, structure-antimalarial relationship analysis of over 40 novel aryl isonitrile compounds has established the importance of the isonitrile functionality as an important moiety for antimalarial activity. Of the many isonitrile compounds exhibiting potent antimalarial activity, two have emerged as leads with activity comparable to that of Artemisinin. The SAR details presented in this study will prove essential for the development new aryl isonitrile analogues to advance them to the next step in the antimalarial drug discovery process.

17-nor-Excelsinidine, a zwitterion monoterpene indole alkaloid isolated from Alstonia scholaris is a subject of synthetic scrutiny. This is primarily due to its intriguing chemical structure which includes a bridged bicyclic ammonium moiety, and its anti-adenovirus and anti-HSV activity. Herein we describe a six-step total synthesis of (±)-17-nor-Excelsinidine from tryptamine. Key to the success of this synthesis is the use of palladium-catalyzed carbonylative heck lactamization methodology which built the 6, 7-membered ring lactam in one step. The resulting pentacyclic product, beyond facilitating the easy access to (±)-17-nor-Excelsinidine, could also serve as a precursor to other related indole alkaloids.