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DB75, a Novel Trypanocidal Agent, Disrupts Mitochondrial Function in Saccharomyces cerevisiae. Charlotte A. Lanteri, 2004.The aromatic diamidines represent a class of compounds with broad-spectrum antimicrobial activity; however, their development is hindered by a lack of understanding of their mechanism of antimicrobial action . DB75 [2,5-bis(4-amidinophenyl)furan] is a trypanocidal aromatic diamidine that was originally developed as a structural analogue of the antitrypanosomal agent pentamidine . DB289, a novel orally active prodrug of DB75, is undergoing phase IIb clinical trials for early-stage human African trypanosomiasis, Pneumocystis jiroveci carinii pneumonia, and malaria . The purpose of this study was to investigate mechanisms of action of DB75 using Saccharomyces cerevisiae as a model organism . The results of this investigation suggest that DB75 inhibits mitochondrial function . Yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75 . DB75 localizes (by fluorescence) within the mitochondria of living yeast cells and collapses the mitochondrial membrane potential in isolated yeast mitochondria . Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration . We conclude that the mitochondrion is a cellular target of DB75 in yeast cells and anticipate that the results of this study will aid in the target-based design of new antimicrobial aromatic diamidines . Structure of a Coenzyme A Pyrophosphatase from Deinococcus radiodurans: a Member of the Nudix Family. Lin-Woo Kang, 2003.Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA) . Nudix enzymes with the same specificity have been found in yeast, humans, and mice . The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily . The structure of the complex of the enzyme with Mg2+, its activating cation, reveals the position of the catalytic site . A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding . Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data . Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard. Burkhard Malorny, 2003.As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific for Salmonella species were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43 Salmonella spp . and 47 non-Salmonella strains . The most selective primer set was found to be 139-141 (K . Rahn, S . A . De Grandis, R . C . Clarke, S . A . McEwen, J . E . Galán, C . Ginocchio, R . Curtiss III, and C . L . Gyles, Mol . Cell . Probes 6:271-279, 1992), which targets the invA gene . An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for the invA primer set . To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with the invA target gene, was constructed . In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 104 CFU/ml was used as the template in the PCR (50 CFU per reaction) . The primer set was further validated in an international collaborative study that included 16 participating laboratories . Analysis with 28 coded ("blind") DNA samples revealed an analytical accuracy of 98% . Thus, a simple PCR assay that is specific for Salmonella spp . and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard . This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data . Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization. Gabriel Zwart, 2003.The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups . We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters . The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes . The optimized assay was made stringent to discriminate at approximately the single-mismatch level . This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database . Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples . Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes . Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes . Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs .
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