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The Antiparasitic Compound Licochalcone A Is a Potent Echinocytogenic Agent That Modifies the Erythrocyte Membrane in the Concentration Range Where Antiplasmodial Activity Is Observed. Hanne L. Ziegler, 2004.The well-known antiparasitic compound licochalcone A is a potent membrane-active agent that transforms normal erythrocytes into echinocytes in parallel with the inhibition of growth of Plasmodium falciparum cultures, the in vitro antiplasmodial effect apparently being an indirect effect on the host cell . In vitro experiments with synchronous cultures demonstrate that inhibition of invasion is the principal mechanism of growth inhibition . The erythrocyte membrane-modifying effect was also transiently observed in vivo in mice after intravenous administration . Burkholderia thailandensis E125 Harbors a Temperate Bacteriophage Specific for Burkholderia mallei. Donald E. Woods, 2002.Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei . We found that B . thailandensis E125 spontaneously produced a bacteriophage, termed  E125, which formed turbid plaques in top agar containing B . mallei ATCC 23344 . We examined the host range of
E125 and found that it formed plaques on B . mallei but not on any other bacterial species tested, including B . thailandensis and B . pseudomallei . Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail . B . mallei NCTC 120 and B . mallei DB110795 were resistant to infection with
E125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively . wbiE was provided in trans on a broad-host-range plasmid to B . mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to
E125 . The 53,373-bp
E125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene . While the overall genetic organization of the
E125 genome was similar to
 -like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes . The
E125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine . The results presented here demonstrate that
E125 is a new member of the
supergroup of Siphoviridae that may be useful as a diagnostic tool for B . mallei .
Role for radA/sms in Recombination Intermediate Processing in Escherichia coli. Cynthia E. Beam, 2002.RadA/Sms is a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and Lon protease . We examined mutations in the radA/sms gene of Escherichia coli for effects on conjugational recombination and sensitivity to DNA-damaging agents, including UV irradiation, methyl methanesulfonate (MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea (HU) . Null mutants of radA were modestly sensitive to the DNA-methylating agent MMS and to the DNA strand breakage agent phleomycin, with conjugational recombination decreased two- to threefold . We combined a radA mutation with other mutations in recombination genes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC . A radA mutation was strongly synergistic with the recG Holliday junction helicase mutation, producing profound sensitivity to all DNA-damaging agents tested . Lesser synergy was noted between a mutation in radA and recJ, recQ, ruvA, ruvC, and recA for sensitivity to various genotoxins . For survival after peroxide and HU exposure, a radA mutation surprisingly suppressed the sensitivity of recA and recB mutants, suggesting that RadA may convert some forms of damage into lethal intermediates in the absence of these functions . Loss of radA enhanced the conjugational recombination deficiency conferred by mutations in Holliday junction-processing function genes, recG, ruvA, and ruvC . A radA recG ruv triple mutant had severe recombinational defects, to the low level exhibited by recA mutants . These results establish a role for RadA/Sms in recombination and recombinational repair, most likely involving the stabilization or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC . Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase. Nanne M. Kamerbeek, 2003.The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester . Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase . On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO . In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides . Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO . To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied . The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer . Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3] . Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%) . The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications . Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil. Richard J. Ellis, 2003.In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis . However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities . To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches . There were small differences in microscopic epifluorescence bacterial counts . Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar . A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies . However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations . The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates . Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample . DGGE bands from both culture-based and culture-independent approaches were sequenced and compared . These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered . Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches .
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