In Vivo Interaction Between Health Food Supplements and Chemotherapeutic Agents on DNA Damage and Cell Production

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.As expected, both Ara-C and MMS treated mice had DNA and cellular damage. SW, FL, Soy and RM10 were found to cause alterations of DNA and/or cellular damage. SW affected to increase genotoxicity of bone marrow while the increased percentage of reticulocytes was observed. FL and RM10 were found to interact with Ara-C. Either the interaction of FL or RM10 with Ara-C resulted in decreased genotoxicity to bone marrow cells. Soy affected to decrease genotoxicity of erythocytes. These results demonstrate that over-the-counter health supplements can alter of DNA and cause cell damage and can interact with chemotherapeutic agents, and that flow cytometric analysis is a rapid and reliable technique for screening the cytotoxic and genotoxic effects of these products.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

In Vivo Interaction Between Health Food Supplements and Chemotherapeutic Agents on DNA Damage and Cell Production

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.As expected, both Ara-C and MMS treated mice had DNA and cellular damage. SW, FL, Soy and RM10 were found to cause alterations of DNA and/or cellular damage. SW affected to increase genotoxicity of bone marrow while the increased percentage of reticulocytes was observed. FL and RM10 were found to interact with Ara-C. Either the interaction of FL or RM10 with Ara-C resulted in decreased genotoxicity to bone marrow cells. Soy affected to decrease genotoxicity of erythocytes. These results demonstrate that over-the-counter health supplements can alter of DNA and cause cell damage and can interact with chemotherapeutic agents, and that flow cytometric analysis is a rapid and reliable technique for screening the cytotoxic and genotoxic effects of these products.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

A novel Rickettsia, Candidatus Rickettsia takensis, and the first record of Candidatus Rickettsia laoensis in Dermacentor from Northwestern Thailand

Abstract Three hundred and forty-four tick samples were collected from vegetation at Taksin Maharat National Park, Tak province, northwestern Thailand. They were morphologically identified and molecularly confirmed by 16S rRNA and COI genes as Dermacentor laothaiensis (n = 105), D. steini (n = 139), and D. auratus (n = 100). These ticks were examined for the spotted fever group rickettsiae (SFGRs) using PCR and DNA sequencing of six genes; 17-kDa, gltA, 16S rRNA, ompA, ompB, and sca4. Of these ticks, 6.10% (21/344) gave positive results for the presence of SFGRs. Phylogenetic analyses of the SFGRs clearly indicated that a novel genotype assigned as Candidatus Rickettsia takensis was detected in D. laothaiensis (19/105) and at lesser frequency in D. steini (1/139). Furthermore, Candidatus Rickettsia laoensis was also found at a low frequency in D. auratus (1/100), the first record in Thailand. Although, the pathogenicities of these SFGRs remain unknown, our findings suggest potential risks of SFGRs being transmitted via ticks near the border between Thailand and Myanmar, a gateway of daily migrations of local people and visitors both legal and illegal

Coxiella-like bacteria in fowl ticks from Thailand

Abstract Background Coxiella bacteria were identified from various tick species across the world. Q fever is a zoonotic disease caused by the bacteria Coxiella burnetii that most commonly infects a variety of mammals. Non-mammalian hosts, such as birds, have also been reported to be infected with the pathogenic form of “Candidatus Coxiella avium”. This research increases the list of tick species that have been found with Coxiella-like bacteria in Thailand. Methods A total of 69 ticks were collected from 27 domestic fowl (Gallus gallus domesticus), 2 jungle fowl (Gallus gallus) and 3 Siamese firebacks (Lophura diardi) at 10 locations (provinces) in Thailand. Ticks were identified and PCR was used to amplify Coxiella bacteria 16S rRNA, groEL and rpoB genes from the extracted tick DNA. MEGA6 was used to construct phylogenetic trees via a Maximum Likelihood method. Results The phylogenetic analysis based on the 16S rRNA gene showed that the Coxiella sequences detected in this study grouped in the same clade with Coxiella sequences from the same tick genus (or species) reported previously. In contrast, rpoB gene of the Coxiella bacteria detected in this study did not cluster together with the same tick genus reported previously. Instead, they clustered by geographical distribution (Thai cluster and Malaysian cluster). In addition, phylogenetic analysis of the groEL gene (the chaperonin family) showed that all Coxiella bacteria found in this study were grouped in the same clade (three sister groups). Conclusions To our knowledge, we found for the first time rpoB genes of Coxiella-like bacteria in Haemaphysalis wellingtoni ticks forming two distinct clades by phylogenetic analysis. This may be indicative of a horizontal gene transfer event

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes

Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation.

Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ΔgshA, ΔgshB, and double ΔgshAΔgshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ΔgshA, ΔgshB and double ΔgshAΔgshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa

Determination of paraquat resistance levels in <i>P</i>. <i>aeruginosa</i> strains.

<p>(A) Paraquat resistance levels in PAO1 containing the mnin-Tn7 vector control (PAO1::Tn7T, red) and Δ<i>finR</i> mutants containing Tn7T (dotted green), Tn7T-<i>finR</i> (purple), Tn7T-<i>fprA</i> (dotted blue), Tn7T-<i>fprB</i> (yellow), or Tn7T-<i>fdx1</i> (dotted black) were determined using plate sensitivity assays. (B) Paraquat (150 μM) resistance levels of <i>P</i>. <i>aeruginosa</i> strains were determined using LB with and without 1% (w/v) KNO₃ supplementation and incubated under aerobic and anaerobic atmospheres. The survival is expressed as a percentage of the CFU on LB plates containing paraquat over the CFU on plates without paraquat. Data shown are means ± SD from three independent experiments.</p

Characterization and binding of purified FinR to the <i>finR-fprA</i> promoter.

<p>(A) Nucleotide sequence showing the <i>finR</i>-<i>fprA</i> promoter structure. +1 indicates the transcriptional start site, and the bold sequences are the putative -35 and -10 promoter motifs. <b><i>CAT</i></b> and <b><i>ATG</i></b> are the translational start codons of FinR and FprA, respectively. The box shaded gray represents the proposed FinR binding site. (B), (C), and (D) Electrophoretic mobility shift assay using purified FinR. A ³²P –labeled DNA fragment (B), mutagenized MU1 and MU2 fragments (C), or the promoter fragments (EBI61-62), with (EBI 61–70) and without (EBI 62–69) proposed FinR binding site (D) spanning the <i>finR-fprA</i> promoter was incubated with increasing amounts of FinR. BSA represents an unrelated protein (2.5 μg BSA). CP and UD signify the cold probe (100 ng unlabeled promoter fragment) and unrelated DNA (1 μg of pUC18 plasmid), respectively, that were added to the binding reaction mixture containing 100 nM FinR. F and B indicate free and bound probes, respectively.</p

Expression analysis <i>finR</i> and <i>fprA</i> in response to various stresses.

<p>The expression levels of <i>finR</i> (A) and <i>fprA</i> (B) were determined using real-time RT-PCR. Exponential-phase cultures of <i>P</i>. <i>aeruginosa</i> PAO1 were subjected to various stress conditions, including 1 mM H₂O₂, superoxide anion-generating agents (0.5 mM plumbagin [PB], 0.5 mM menadione [MD] and 0.5 paraquat [PQ]), organic hydroperoxides (1 mM cumene hydroperoxide [CHP] and 1 mM <i>t</i>-butyl hydroperoxide [tBH]), 1 mM 2,2’-dipyridyl (DIPY), high salts (0.5 M NaCl and 0.5 M KCl), or 0.04% NaOCl for 15 minutes prior to RNA preparation for real-time RT-PCR analysis. Relative expression was analyzed using the 16S rRNA gene as the normalizing gene and was expressed as the fold expression relative to the level of uninduced (UN) PAO1. Data shown are means ± SD of three independent experiments.</p