Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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The main research of this thesis includes three sections. The purpose of first part is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 8 macrolide antibiotics and lincosamides inside fish tissue, including erythromycin (ERM), oleandomycin (OLD), kitasamycin (KIT), tylosin (TYL), josamycin (JOS), spiramycin (SPM), tilmicosin (TIL), and lincomycin (LIN). Homogenized samples are first extracted with acetonitrile, dehydrated with sodium sulphate anhydrous, and then condensed. After the residue was redissolved in methanol and the extracts were partitioned with n-hexane to remove lipids, the sample is filterced and detected by LC/MS-MS using chromatography columns of Agilent HC-C18 (5£gm, 150 mm ¡Ñ4.6 mm). The mobile phase A was 5mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was acetonitrile. The analysis of 8 macrolide antibiotics and lincosamides can be achieved within 10 minutes with electrospray ionization-tandem mass spectrometry in positive mode using multiple reaction monitoring (MRM) for simultaneous detection. The second part is to verify the method by regulation of European Union (EU) resolution scheme (2002/657/EC). In the case where the drug is set as allowed drug, the recovery rate under gradient addition according to MRL is between 93.64% to 106.67%, and the CV is between 0.27% to 7.17%. In the case where the drug is set as prohibited drug, the recovery rate under gradient addition according to MRPL is between 96.35%~104.88%, and the CV is between 6.77%~13.91%. As a result, the decision limit (CC£\) and the Detection capability (CC£]) of the 8 macrolide antibiotics and lincosamides is between 0.24 to 0.40£gg kg-1 and 0.33 to 0.49£gg kg-1. The last section is to evaluate the stability of drugs in fish body under domestic preservation and process methods on fish, including refrigeration at -20¢J and cold storage at 4 ¢J. The test is implemented by adding the drug into fish tissue according to MRL and detecting the antibiotics residue after regulated 40 days. Besides, the effect on activity of drug residue in fish body after boiling at 100 ¢J is compared. The results show that the residual amount of spiramycin, josamycin, tilmicosin, and lincomycin is below 35% while that of erythromycin, oleandomycin, kitasamycin, and tylosin will be below 20%. Therefore, the drugs including erythromycin, josamycin, tylosin, and lincomycin will stay stably in fish tissue if they are stored under -20 ¢J. However, it may affect human health if the fish contains such antibiotic residues is not boiled.

From an agronomic perspective, the spreading of manure (sometimes containing antibiotics) onto agricultural fields is beneficial to the soil as a renewable source of fertilizer by increasing organic matter and providing nutrient inputs for crops. However, the use of antibiotics can be excessive, resulting in manures containing residual antibiotics contaminating soils and waterways. Thus, there is a need to improve existing or develop new management practices to minimize the losses of antibiotics from manure entering waterways and groundwater. Biochar is a carbon-rich material produced from the oxygen-free pyrolysis of biomass. Generally, biochars have high surface area and sorb organic compounds and trace metals; thus, it is reasonable to hypothesize that biochars sorb antibiotics. The main goal of this research was to investigate if incorporated biochar to soil facilitates the transport of antibiotics under simulated rainstorm events. The specific objectives were to investigate the losses of surface-applied antibiotics to soils with different (1) application rates of biochar and rainfall intensities, and (2) if the losses were antibiotic type-dependent. <br>

Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...

Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...

4-Alkyl-L-proline derivatives (APD) are specialized precursors involved in the biosynthesis of at least three groups of different natural compounds: some pyrrolo-1,4-benzodiazepines with antitumor activity, bacterial hormone hormaomycin and clinically used lincosamide antibiotic lincomycin. These compounds share a biosynthetic pathway encoded by 5 or 6 homologous genes present in the biosynthetic gene clusters of the producing organisms. Similarities in biosynthesis and differences between APD structures of these compounds could be used to prepare a hybrid producing strain of biologically more effective lincomycin derivative. Unusual amino acid 4-propyl-L-proline (PPL) is the APD precursor of lincomycin. The originally proposed scheme of the PPL pathway does not comply with our current knowledge. Therefore, it was necessary to revise this scheme according to new results. The first two steps of the PPL pathway are functionally proved. Probing the next step was the main aim of this work. The protein LmbW was overproduced and its methyltransferase activity was confirmed in vitro. LmbW is able to directly methylate intermediate of second step of the pathway while the originally scheme proposed methylation at a later stage of biosynthesis. LmbW is also able to attach a longer alkyl chain to its substrate. This...

The genus Streptomyces produces more than a half of the known bioactive substances, ranking it among the most important bacterial taxons. Streptomyces lincolnensis ATCC 25466 encodes a biosynthetic gene cluster for lincomycin biosynthesis in its genome. Apart from the biosynthetic and regulatory genes, the cluster also contains three resistance genes, lmr(A), lmr(B) a lmr(C), which could protect of the host from the toxicity of a synthesized antibiotic. The Lmr(C) protein belongs to ARE proteins which generaly confer resistance to clinically important classes of antibiotics: macrolides, streptogramins, lincosamides and pleuromutilins. In addition to antibiotic producers, ARE proteins are also present in pathogenic microorganisms. However, the resistance mechanism conferred by these protins which belong to ABC transporters, even though they lack the transmembrane domain, have not been characterized yet. This makes the ARE proteins an interesting subject of the research. Using deletion mutants in resistance genes lmr(A), lmr(B) a lmr(C) we studied their effect on the lincomycin production and resistance to lincosamides, lincomycin and clindamycin with special focus on the function of the lmr(C). We have found that deletion of lmr(C) does not significantly influence lincomycin production and...

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Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...