Call number: LD2668 .T4 1986 H87 / Master of Science / Clinical Sciences

Orientador: Maria Del Pilar Taboada Sotomayor / Banca: Marcos Roberto de Vasconcelos Lanza / Banca: André Luiz dos Santos / Resumo: O presente trabalho mostra o desenvolvimento do primeiro biossensor descrito na literatura para determinação de antibióticos β-lactâmicos, usando pasta de carbono modificada com a enzima β-lactamase, e sua aplicação em amostras de leite de vaca in natura. A partir de estudos prévios de otimização da preparação do biossensor, o grafite em pó foi ativado com carbodiimida para permitir a ligação covalente da enzima β-lactamase que, junto com o mediador de elétrons ftalocianina de cobalto ofereceram variações na corrente catódica na presença da penicilina G, sendo esta escolhida para representar os antibióticos β-lactâmicos. Utilizando este procedimento para a construção dos biossensores, foi possível registrar voltamogramas cíclicos e amperogramas que permitiram quantificar a benzilpenicilina (penicilina G) em condições de análise também otimizadas, que incluíram estudo da quantidade de mediador na pasta; o pH, tipo e concentração do eletrólito usado para realização das medidas e o potencial aplicado na amperometria. Com o método otimizado foi possível detectar penicilina G em amostras de leite in natura, fortificadas com o antibiótico, com alta exatidão (erro relativo de 1%) e boa precisão (desvio padrão relativo de 8,3%, n = 3), mostrando que o biossensor desenvolvido é uma ferramenta promissora para detecção de penicilinas, e que ao serem realizados mais estudos para diminuir seu limite de quantificação, poderá se tornar uma um método de análise alternativo aos kits comerciais existentes / Abstract: This work describes the development of the first biosensor described in the literature for the determination of β-lactam antibiotics using a carbon paste electrode modified with the enzyme β-lactamase, and its application in samples of fresh cow's milk. After optimizing the biosensor preparation procedure, the graphite powder was activated with carbodiimide to allow covalent binding of the enzyme β-lactamase, which together with the electron mediator, cobalt phthalocyanine, offered variations in cathodic currents in the presence of penicillin G, which was chosen as representative of the β-lactam antibiotics. Using this procedure for the construction of biosensors, it was possible to record cyclic voltammograms and amperograms that enabled quantification of benzylpenicillin (penicillin G) under analytical conditions that had been optimized in terms of the amount of mediator in the paste, the pH, the type and concentration of the electrolyte used in the measurements, and the applied amperometric potential. With the optimized method, it was possible to detect penicillin G in samples of fresh milk fortified with the antibiotic, with high accuracy (error of 1%) and adequate precision (RSD of 8.3%, n=3), demonstrating that the proposed biosensor is a promising tool for the detection of penicillins. Once quantitation limits have been further improved, the analytical method could become an alternative to existing commercial kits / Mestre

Benzylpenicilloyl-poly-L-cysteine is prepared by reacting benzylpenicillenic acid with poly-L-cysteine at 370° in water at pH 8.3 or buffer at pH 7.98. The preparations of penicilloyl-cysteine, S-acetamidomethyl-polycysteine, and S-acetamidomethyl-penicilloyl-polycysteine are also described. C14-labeling and penamaldate assays are used to determine penicilloyl content. The iodoacetic acid method is used to ascertain thiol content.The ultraviolet spectra for penicilloyl-polycysteine, pencilloyl-cysteine, poly-L-cysteine, poly-S-carbobenzoxy-L-cysteine, S-acetamidomethyl-polycysteine and S-acetamidomethyl-penicilloyl-polycysteine are reported. The infrared spectra of penicilloyl-polycysteine, penicilloyl-cysteine, poly-S-carbobenzoxy-L-cysteine, S-acetamidomethyl-polycysteine, and S-acetamidomethyl-penicilloyl-polycysteine are reported. The nmr spectra of poly-L-cysteine, penicilloyl-cysteine, S-acetamidomethyl-polycysteine, and S-acetamidomethyl-penicilloyl-polycysteine are discussed.The kinetic rates of reaction of benzylpenicillenic acid at 37.5 ± 0.50 in buffer, cysteine, N, S-di-CBZ L-cysteine, poly-L-cysteine, poly-S-CBZ L-cysteine andβ-mercaptoethylamine are compared.Ball State UniversityMuncie, IN 47306

Benzylpenicillin, a-aminobenzylpenicillin, phenoxymethylpenicillin, and 2,6-dimethoxy-phenylpenicillin were incubated with whole human blood. Migration patterns into human erythrocytes were compared spectrophotometrically.A possible inhibition effect by chloroquine on benzyl-[14C]-penicillin in red blood cells was studied via a Beckman LS-100C Liquid Scintillation Counter.An intraerythrocytic protein, carbonic anhydrase, was investigated as being responsible for the hydrolysis of benzylpenicillin. Three carbonic anhydrases (a, b, and c) were quantitatively measured via a Beckman IR 4250 Spectrophotometer for their effect upon the a-lactam ring of benzylpenicillin.Ball State UniversityMuncie, IN 47306

Benzylpenicillin was found to migrate irreversibly into human erythrocytes. No detectable amounts of penicillin bind to the erythrocyte membrane. After migration into the cell, benzylpenicillin is hydrolyzed into benzylpenicilloic acid, which can not migrate out of the cell. DNDS did not affect the appearance of benzylpenicilloic acid in the erythrocyte, therefore showing that: 1) benzylpenicillin does not hydrolyze to benzylpenicilloic acid in the incubation mixture then migrate to the cell, 2) benzylpenicillin migrates into the erythrocyte and is hydrolyzed to benzylpenicilloic acid. Benzylpenicilloic acid does not migrate into erythrocytes.Ball State UniversityMuncie, IN 47306

N-Thiolated beta-lactams are a new class of anti-MRSA and anti-Bacillus agents that have recently been reported by our laboratories. From previous studies performed in our laboratories, it is believed that the N-thiolated beta-lactams exert their antimicrobial activity through a unique mode of action that is completely unlike that of classical beta-lactam antibiotics. In the first chapter of this dissertation, a review of previously prepared N-thiolated beta-lactam analogues and their mode of action is presented. In the second chapter, the synthesis of seven different C3-oxygenated derivatives is described. These analogues were tested for antibacterial activity against Staphylococcus aureus, nine different strains of MRSA, and seven different species of Bacillus. The results of the antibacterial testing will be discussed in relation to the differences in the structures of the analogues. In chapter 3, the design and synthesis of two new, functionalized caprolactone monomers are presented. FTSThese monomers were subsequently cooligomerized with epsilon-caprolactone, as described in chapter 4. N-thiolated beta-lactams were attached to the functionalized oligomers. These antibiotic containing oligomers were then screened for activity against MSSA, MRSA, and Bacillus. The results of these biological tests and their implications for future experiments are discussed.

The goal of our research was to optimize the conditions of chromatographic analysis of penicillin, to define minimal found analysis concentration, analyse contaminated milk samples with given certain concentration and establish resumption of penicillin G. Therefore, we aimed at analysing milk samples‘ contamination with penicillin of randomly selected cows from study farms. The research was performed in Lithuanian Academy of Veterinary which has a practical training and experiment centre in Muniskes cow farm, state enterprise „Pieno tyrimai“ and Food Institute of Kaunas Technological University. State enterprise „Pieno tyrimai“ used preparation LPT to define milk inhibitors. After the bacteriological milk samples analyses 8 out of 25 samples were found to portray remnants of inhibitory materials. Optimal chromatographic conditions were established by the use of experiment, in accordance with a thorough analysis of literature concentrating on chromatographic methods dealing with penicillin G determination. The following are defined conditions for the chromatographic examination of penicillin G: composition of a mobile phaze 0 – 10 min 5% ACN / 95% 0,125 M phosphate buffer (pH=3,5), 12-22 min 70 % ACN / 30% 0,125 M phosphate buffer, 24-30 min 5% ACN / 95% 0,125 M phosphate buffer (gradient flow mode). Period of assay suspention was about 17 minutes. Resumption of contaminated milk samples with penicillin G 500 mg/kg, 250 mg/kg, 50 mg/kg and 4 mg/kg varied from 45 to 76 %. The... [to full text]

This thesis describes an investigation of the mechanism of the bifunctional, a-ketoglutarate dependent dioxygenase, deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of this to deacetylcephalosporin C (DAC). The conversion of the unnatural substrate 3-exomethylene cephalosporin C by DAOC/DACS has been investigated in detail. A new metabolite was isolated from incubations of the deuterated [4-²H]-3-exomethylene cephalosporin C, and was identified as the 3β-spiroepoxide cepham, (2Ṟ,3Ṟ,6Ṟ,7Ṟ)-l-aza-[2-²H]-3-spiroepoxy-7-[(5Ṟ)-5-amino- 5-carboxypentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylic acid. The results obtained indicate that this metabolite is a shunt product whose formation is enhanced by the operation of a deuterium kinetic isotope effect on an enzyme-bound intermediate. It has also been found that this 3β-spiroepoxide cepham is further converted by DAOC/DACS to 3-formyl cephalosporoate products. The mechanism of oxygenation of DAOC/DACS was investigated through ¹⁸O-labelling studies. Incubations of [2-¹³C,3-²H]penicillin N and [4-²H]-3-exomethylene cephalosporin C with DAOC/DACS were carried out under ¹⁸O₂ or in H₂¹⁸O. Incorporation of ¹⁸O-label into the products [3-¹³C]DAC, [3-¹³C,4-²H]-3β-hydroxycepham and 3β-spiroepoxide cepham was observed from both sources. The results suggest that intermediates capable of oxygen-exchange are formed during the enzymatic reactions. Two substrate analogues, the 5-epipenicillin N and the 2β-difluoromethyl penicillin N, have been synthesised in order to probe the substrate specificity of DAOC/DACS with respect to the ring-expansion activity. The 5-epipenicillin N was not accepted as a substrate by DAOC/DACS, and the observations made indicate that it was unstable under the incubation conditions. No product was either observed from incubations of the 2β-difluoromethyl penicillin N with DAOC/DACS, although bioassay tests suggested a cephem product had been formed in very small amounts. Finally, the results of a substrate specificity comparison between the soluble recombinant enzymes deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS) from Cephalosporium acremonium and deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus are described.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Asim K. Bej, Susan K. Hollingshead, Daniel D. Jones, Stephen A. Watts. Description based on contents viewed June 23, 2009; title from PDF t.p. Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 1947.