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Synergism between Amodiaquine and Its Major Metabolite, Desethylamodiaquine, against Plasmodium falciparum In Vitro. S. T. Mariga, 2004.The in vitro activity of the prodrug amodiaquine and its metabolite monodesethyl-amodiaquine has been studied for three strains of Plasmodium falciparum: LS-2, LS-3, and LS-1 . Both compounds showed significant activity against all three strains; the activity of amodiaquine was slightly higher than that of the metabolite . By use of a checkerboard design, interaction studies with both compounds yielded evidence of significant synergism; means of the sums of the fractional inhibitory concentrations were 0.0392 to 0.0746 for strain LS-2, 0.1567 to 0.3102 for strain LS-3, and 0.025 to 0.3369 for strain LS-1 . In further investigations, the interaction of amodiaquine with monodesethyl-amodiaquine was tested at clinically relevant concentrations of both compounds . In these studies, involving amodiaquine at picomolar and femtomolar concentrations, the compound was found to exert high potentiating activity on monodesethyl-amodiaquine . This interaction produced mean ratios of observed to expected activity of 0.0505 to 0.0642 for strain LS-2, 0.0882 to 0.3820 for strain LS-3, and 0.0752 to 0.2924 for strain LS-1 . The synergistic activity was most marked at monodesethyl-amodiaquine/amodiaquine ratios up to 100,000:1 but was still evident at higher ratios . Sample Size, Library Composition, and Genotypic Diversity among Natural Populations of Escherichia coli from Different Animals Influence Accuracy of Determining Sources of Fecal Pollution. LeeAnn K. Johnson, 2004.A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli . Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E . coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep . HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting . Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification . However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification . The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm . Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E . coli was very great and is most likely accounting for our inability to correctly classify many environmental E . coli isolates . Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods . Characterization of Two Noncellulosomal Subunits, ArfA and BgaA, from Clostridium cellulovorans That Cooperate with the Cellulosome in Plant Cell Wall Degradation. Akihiko Kosugi, 2002.Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases . To characterize the  -L-arabinosidases that are involved in hemicellulose degradation, we screened the C . cellulovorans genomic library for clones with
-L-arabinofuranosidase or
-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated . The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci . The amino acid sequences for ArfA and BgaA were related to
-L-arabinofuranosidase and ß-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively . Recombinant ArfA (rArfA) had high activity for p-nitrophenyl
-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl
-L-arabinopyranoside . On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl
-L-arabinopyranoside but also p-nitrophenyl ß-D-galactopyranoside . However, when the affinities of rBgaA for p-nitrophenyl
-L-arabinopyranoside and p-nitrophenyl ß-D-galactopyranoside were compared, the Km values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for
-L-arabinopyranose residues than for ß-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 ß-galactosidase . Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction . When rArfA and rBgaA were incubated with ß-1,4-xylanase A (XynA), a cellulosomal enzyme from C . cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone . These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits .
Biodegradation of an Alicyclic Hydrocarbon by a Sulfate-Reducing Enrichment from a Gas Condensate-Contaminated Aquifer. Luis A. Rios-Hernandez, 2003.We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer . The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced . During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles . Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid . Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon . Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer . However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill . Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene . Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group . The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments . Unusual Organization for Lactose and Galactose Gene Clusters in Lactobacillus helveticus. Maria Grazia Fortina, 2003.The nucleotide sequences of the Lactobacillus helveticus lactose utilization genes were determined, and these genes were located and oriented relative to one another . The lacLM genes (encoding the ß-galactosidase protein) were in a divergent orientation compared to lacR (regulatory gene) and lacS (lactose transporter) . Downstream from lacM was an open reading frame (galE) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as lacM . The lacR gene was separated from the downstream lacS gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (lacS') . Northern blot analysis revealed that lacL, lacM, and galE made up an operon that was transcribed in the presence of lactose from an upstream lacL promoter . The inducible genes lacL and lacM were regulated at the transcriptional level by the LacR repressor . In the presence of glucose and galactose galE was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively . Lactose transport was inducible by addition of lactose to the growth medium .
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