Thesis (Ph.D.)--Baylor University, 2005. / Includes bibliographical references (p. 259-273).

The prevention and control of salmonellae in commercial swine operations are becoming increasingly important. The current approach focuses on identifying sources and/or origins of salmonellae contamination before swine are processed for human consumption. The objective of the current study was to assess strain variability among salmonellae grouped by serotype and to determine common origins of contamination (farm or slaughter plant). Salmonellae were previously collected from swine at slaughter, serotyped by the National Veterinary Services Laboratory and stored at - 70??C. Pulsed-field gel electrophoresis (PFGE) was performed to genotypically characterize serotypic isolates using restriction endonuclease XbaI. Dendrogram comparisons were also used to assess genotypic similarity when multiple genotypes existed. This study found PFGE to be more discriminatory than serotyping indicating that multiple genotypic strains existed among selected serotypes. On the basis of PFGE results alone, origins of contamination could not be determined in this study. It is suggested by the author, that origins of contamination could be further defined pending future research, in which in-depth longitudinal studies are included. When used as an adjunct to conventional typing methods, PFGE may prove to be a substantial subtyping system in epidemiologic investigations to identify point-of-entry contaminants to the food chain.

In the first part of this work, magnetized nanoporous silica based microparticles (MMPs) are used for horseradish peroxidase (HRP) immobilization and applied in amperometric peroxidase-based biosensors. A homemade magnetized carbon paste electrode permits the MMPs attraction close to the electrode surface. The resulting original biosensor is applied to the investigation of enzymatic oxidation of model drug compounds namely, clozapine (CLZ) and acetaminophen (APAP) by HRP in the presence of hydrogen peroxide. The biosensor operates at a low applied potential and the signal corresponds to the electro-reduction of electroactive species enzymatically generated. The biosensor allows performing the quantitation of the two drug compounds in the micromolar concentration range. It allows also the study of thiol compounds based on the inhibition of the biosensor response. Interestingly, distinct inhibition results are observed for HRP entrapped in the silica microparticles compared to the soluble HRP. We expect that this type of biosensors holds high promise in quantitative analysis and in biotransformation studies of drug compounds. In the second part of this thesis work, HRP immobilized magnetic nanoparticles are injected on-line and magnetically retained, as a microreactor, in the capillary of a CE setup. The purpose of such a configuration is to develop an analytical tool for studying “in vitro” drug biotransformation. The advantages expected are (i) minimum sample (drug compound) and biocomponent (enzyme) consumption, (ii) high analysis throughput, (iii) selectivity and sensitivity. In order to illustrate the potential of such an instrumental configuration, it has been applied to study acetaminophen as model drug compound. The mechanistic information obtained by the HRP/H2O2 system is in agreement with literature data on acetaminophen metabolization. Horseradish peroxidase kinetic studies are realized by this setup and the apparent Michaelis constant is determined. Capillary electrophoresis permitted the identification of APAP off-line biotransformed products such as N-acetyl-p-benzoquinone imine (NAPQI), the APAP dimer and APAP polymers as inferred from literature data. The formation of the APAP dimer was further confirmed by electrospray ionization mass spectrometry.

Tissue factor (TF), a 47 kDa glycoprotein, is the initiator of the extrinsic pathway of blood coagulation and consequently of the upmost importance when damage to blood vessel occurs. The source of TF in circulation has been investigated. However, the source of TF is still not clear. One theory is that platelets express and increases the expression of TF after stimulation and the aim of our report was to investigate whether platelets really are a source for TF in circulation. Using specific primers for TF mRNA, platelets in plasma from healthy volunteers and from patients suffering from cardiac infarction were analyzed by using real-time polymerase chain reaction (PCR). Gel electrophoresis was performed after amplification of TF mRNA to verify the results. The samples were negative for TF when using real-time PCR and the few positive all had cycle threshold (Ct) values above 35. The contamination by monocytes was analyzed by using real-time PCR, with primers for CD14 and showed low amounts. After analysis, our conclusion was that platelets do not express TF. Although some samples had positive real-time PCR, the Ct values were all above 35, meaning they had very few transcripts in the initial samples and that the biological importance is uncertain. Since contamination of CD14 positive cells were found in most samples it can’t be ruled out that the origin of the positive TF mRNA is from monocytes.

No description available.

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Deinococcus radiodurans is a highly UV and radio resistant bacterium. The addition of Mn2+ could induce an Mn-CD effect in this bacterium. In this study, we used two-dimensional polyacrylamide gel electrophoresis to compare and analyze the expressed-proteins under various growth conditions, such as temperature and the presence of Mn2+ or not. The results showed that Mn2+ could affect the similarity proteins expression. As the time of Mn-CD effect elapsed longer, the similarity of the proteins from different growth phages became lower. This indicated that Mn2+ indeed could induce or repress the gene expression. From the 2-D gel analysis, there were fourteen proteins had been induced or overexpressed. Five of them were the proteins with the functions for the synthesis and decomposition of proteins and DNA, others were ATP-binding cassette¡]ABC¡^transporter¡Bsuperoxide dismutase［Mn］, and the rest five were the hypothetical proteins with unclear function. In addition, this study also found that the cultivation temperatures caused conformational and physiological modification of the cell. The addition of Mn2+ could enhance the viability of the bacterium at higher temperature.

Determination of lead and iodine species by capillary electrophoresis inductively coupled plasma mass spectrometry.

The prevention and control of salmonellae in commercial swine operations are becoming increasingly important. The current approach focuses on identifying sources and/or origins of salmonellae contamination before swine are processed for human consumption. The objective of the current study was to assess strain variability among salmonellae grouped by serotype and to determine common origins of contamination (farm or slaughter plant). Salmonellae were previously collected from swine at slaughter, serotyped by the National Veterinary Services Laboratory and stored at - 70??C. Pulsed-field gel electrophoresis (PFGE) was performed to genotypically characterize serotypic isolates using restriction endonuclease XbaI. Dendrogram comparisons were also used to assess genotypic similarity when multiple genotypes existed. This study found PFGE to be more discriminatory than serotyping indicating that multiple genotypic strains existed among selected serotypes. On the basis of PFGE results alone, origins of contamination could not be determined in this study. It is suggested by the author, that origins of contamination could be further defined pending future research, in which in-depth longitudinal studies are included. When used as an adjunct to conventional typing methods, PFGE may prove to be a substantial subtyping system in epidemiologic investigations to identify point-of-entry contaminants to the food chain.

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. Today, P. longifolia var. pendula is in large-scale cultivation in south Taiwan as a landscape plant. A new compound, clerodane diterpenoid 16-hydroxycleroda-3,13-dien-15, 16 -olide¡]CD¡^, was isolated from the bark of Polyalthia longifolia var. pendula. The compound was shown with cell growth inhibitory property at first screen in KMU¡]Kaohsiung Medical University¡^. In our study, antiproliferative activity of CD on human colorectal adenocarcinoma cells¡]HT29 and HCT116¡^were tested and the inhibitory concertration of 50¢Mcell viability¡]IC50¡^is 5 £gg/ml¡]HCT116¡^ and 10 £gg/ml¡]HT29¡^determined by MTT(3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The caspase 3 and PARP cleavage experiment results indicated that CD induced apoptosis is dose dependent manner. We found that CD induced sub-G1 accumulation and reactive oxygen species(ROS) release by flow cytometry analysis. Pretreatment of N-Acetyl-L-cystine(NAC), an antioxidant agent, can reverse the anti-proliferation effect by CD. We found that CD induced loss of mitochondria membrane potentiol¡]£G£Zm¡^. We also analysis differentially expressed proteins by 2-D electrophoresis and find drug response proteins, e.g. HSP10, Profilin-1, Peroxindoxin-1. RT-PCR and western blot was performed to confirm the protein expression changes. It is interesting to reveal the role of these proteins in the colorectal cancinogenesis and anti-tumor drug response.