<p> In vivo and in vitro studies on the biosynthesis of vasopressin in the supraoptic nuclei of the dog and guinea pig1 using 35s-cysteine and 3H-tyrosine have suggested that vasopressin could be synthesized by wa;y of a precursor, which is modified to release active hormone. In vivo injection of 3H-tyrosine into the cerebrospinal fluid of rats2 had resulted in incorporation of the label into both oxytocin and vasopressin. In this work, an attempt was made to develop an in vitro system for the biosynthesis of oxytocin. Incubations of either 3H-isoleucine or 14c-leucine with rat hypothalamic neuronal perikaya, ribosomes and cell sap, cell sap, fractions of cell sap and homogenate, and incubations of 3H-isoleucine and l4C-leucine with rat hypothalamic tissue fragments were analyzed for the incorporation of label into purified hormone. Gel filtration, partition chromatography, high voltage electrophoresis, and thin layer chromatography were applied, followed by measurement of radio-activity and biological activity. It is concluded that in no reproducible case was either radioactive isotope incorporated into material with the chromatographic and biological properties of oxytocin. Other radioactive products of incubation were detected in hypothalamic cell sap, ribosomes and cell sap, and homogenate. In hypothalamic homogenate incubations, considerable degradation of both oxytocin and other material absorbing at 280 nm was observed. It is suggested that future investigations should attempt to first develop isolation procedures for the labelled hormone produced in vivo, and then reduce the complexity of the system in small stages, through the cultured hypothalamic-neurohypophyseal system of Sachs3 to simpler in vitro systems. </P> / Thesis / Master of Science (MSc)

The biosynthesis of hydrastine and of berberine was investigated by feeding radioactive tyrosine and dopamine to Hydrastis canadensis L. It was shown that both hydrastine and berberine are specifically derived from two molecules of tyrosine, but that the ratio of incorporation of the two molecules of radiotyrosine was not unity, but varied with time. Dopamine was also incorporated into hydrastine without randomization, but only one molecule was utilized. The results, which are consistent with Robinson’s hypothesis of biogenesis, throw light on some of the individual steps of the biosynthetic path by which hydrastine and berberine are derived. / Thesis / Doctor of Science (PhD)

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No description available.

No description available.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. / Thesis / Doctor of Philosophy (PhD)

<p>The glycopeptide antibiotic vancomycin is used as front line treatment for serious Gram-positive infections and resistance to this drug is widespread. Three genes are essential for resistance, <em>vanHAX</em>, which are controlled by a two-component regulatory system VanR and VanS. Here, glycopeptide resistance is found to be ancient and diverse in the environment. A <em>vanA</em> open reading frame from 30 000 yr old DNA was identified and the enzyme was shown to be as functional as comparable to modern day VanA homologs. In the environment resistance is found to be diverse and widespread. For example, the organism <em>Desulfitobacterium hafniense</em> Y51 VanH was shown as non-essential in conferring inducible resistance. Furthermore in the glycopeptide producer <em>Amycolatopsis balhimycina</em> harboring the classic <em>vanHAX,</em> a functional VanA homolog is described as an orphan gene outside of any recognizable gene cassette .</p> <p>Glycopeptides are natural products made by members of the Actinomycete family and are modified by different types of tailoring enzymes. Of particular interest is the glycopeptide A47934, which is ‘aglyco’, and sulfated. The sulfotransferase StaL will transfer not only a sulfate group to A47934, but a sulfamide and fluorosulfonate group. Focusing on additional tailoring enzymes, the biosynthetic cluster of the sulfated glycopeptide UK68597 was sequenced. This cluster has provided a resource for glycopeptide tailoring enzymes for use to modify the A47934 backbone. Sulfation was the first focus and the substrate promiscuity of StaL was explored to expand the chemical diversity A47934 and vancomycin. This work has led to the discovery that glycopeptide sulfation will antagonize the activation and expression of <em>vanHAX</em>. A new sulfated vancomycin derivative was created with this antagonizing activity in the clinical pathogen <em>Enterococcus faecium</em> of the VanB phenotype. Implications of these results and the further use of tailoring enzymes to modify glycopeptides to antagonize resistance will be discussed.</p> / Doctor of Philosophy (PhD)

Genetic and biochemical studies were conducted on S. toyocaensis NRRL 15009, a gram-positive spomlating filamentous bacterium, and producer of the glycopeptide antibiotic A47934. This compound is structurally similar to the clinically important antibiotic vancomycin, and the recent spread of vancomycin-resistant enterococci (VRE) in North American hospitals has driven the need for new glycopeptides with enhanced activities. Studies were aimed at developing an understanding of the mechanism of A47934 biosynthesis inS. toyocaensis NRRL 15009, as well as the mechanism of resistance employed by this organism. Two cosmid clones, containing a partial A47934 biosynthesis gene cluster on a total of65 kilobases of S. toyocaensis NRRL 15009 chromosomal DNA, were isolated for study. Preliminary sequencing indicates the presence of several genes predicted in glycopeptide assembly, such as peptide synthetases and glycosyltransfe~ases. Furthermore, using a oligonucleotide probe designed to identify D-alanine-D-alanine ligases, an 8.1 kilobase chromosomal fragment was isolated from S. toyocaensis NRRL 15009 and found to contain genes very similar to VRE vanH, vanA and vanX. Phylogenetic analysis of the predicted products of these genes showed them to be more similar to the VRE enzymes than any other in each enzyme class. These genes were also found in the vancomycin producer A. orienta/is C329.2 and several other glycopeptide antibiotic producing organisms. Not only does this imply that these organisms employ a mechanism of resistance similar to clinical VRE, it also suggests that these organisms may have been the source of the VRE genes. The enzymes VanHst and DdlN were studied in some detail and found to have biochemical properties similar to their corresponding VRE enzymes VanH and VanA, respectively. Given that the latter group of enzymes has physical properties that have impeded detailed analysis of enzyme mechanism, these new enzymes could find use as model systems in drug development programs. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24551号 / 薬科博第168号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 竹島 浩, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

AgaR, a protein in <i>Escherichia coli</i> thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for <i>agaR, agaZ</i> and <i>agaS</i>, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which <i>agaR</i> had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. <i>In vivo</i> and <i>in vitro</i> analysis of the <i>agaZ</i> promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the <i>aga</i> promoters was less sensitive to catabolite repression in revertants capable of <i>N</i>-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the <i>E. coli</i> genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of <i>sn</i>-glycerol 3-phosphate metabolism in <i>E. coli</i>, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P_glpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P_glpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of <i>sn</i>-glycerol 3-phosphate in <i>E. coli</i> by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K_m (SSO₃²⁻) and K_m (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate. / Ph. D.