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Terpenes Arrest Parasite Development and Inhibit Biosynthesis of Isoprenoids in Plasmodium falciparum. Herbert Rodrigues Goulart, 2004.Development of new drugs is one of the strategies for malaria control . The biosynthesis of several isoprenoids in Plasmodium falciparum was recently described . Interestingly, some intermediates and final products biosynthesized by this pathway in mammals differ from those biosynthesized in P . falciparum . These facts prompted us to evaluate various terpenes, molecules with a similar chemical structure to the intermediates of the isoprenoids pathway, as potential antimalarial drugs . Different terpenes and S-farnesylthiosalicylic acid were tested on cultures of the intraerythrocytic stages of P . falciparum, and the 50% inhibitory concentrations for each one were found: farnesol, 64 µM; nerolidol, 760 nM; limonene, 1.22 mM; linalool, 0.28 mM; and S-farnesylthiosalicylic acid, 14 µM . All the terpenes tested inhibited dolichol biosynthesis in the trophozoite and schizont stages when [1-(n)-3H]farnesyl pyrophosphate triammonium salt ([3H]FPP) was used as precursor . Farnesol, nerolidol, and linalool showed stronger inhibitory activity on the biosynthesis of the isoprenic side chain of the benzoquinone ring of ubiquinones in the schizont stage . Treatment of schizont stages with S-farnesylthiosalicylic acid led to a decrease in intensity of the band corresponding a p21ras protein . The inhibitory effect of terpenes and S-farnesylthiosalicylic acid on the biosynthesis of both dolichol and the isoprenic side chain of ubiquinones and the isoprenylation of proteins in the intraerythrocytic stages of P . falciparum appears to be specific, because overall protein biosynthesis was not affected . Combinations of some terpenes or S-farnesylthiosalicylic acid tested in this work with other antimalarial drugs, like fosmidomycin, could be a new strategy for the treatment of malaria . The E1ß and E2 Subunits of the Bacillus subtilis Pyruvate Dehydrogenase Complex Are Involved in Regulation of Sporulation. Haichun Gao, 2002.The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1  and E1ß), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH) . There are two promoters: one for the entire operon and an internal one in front of the pdhC gene . The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1 and E1ß . Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1 is essential . The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium . The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level . This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity . Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations . Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III) . This analysis was confirmed by fluorescent membrane staining . The E1ß and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation .
Identification of a Protein That Inactivates the Competence-Stimulating Peptide of Streptococcus pneumoniae. Mathieu Bergé, 2002.Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP) . A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction . By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO . Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation . To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO . Chlorination of Indicator Bacteria and Viruses in Primary Sewage Effluent. Julia A. Tree, 2003.Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents . Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards . It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms . This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite . Seeding experiments showed that inactivation (>5 log10 units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log10 unit) of F+-specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses . However, seeded poliovirus was significantly more susceptible (2.8 log10 units) to inactivation by chlorine than was the FRNA bacteriophage . To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage . Multifactorial analysis of variance showed that there was no significant difference (P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage . However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses (P < 0.001) . This may reflect differences in the way indigenous virus is presented to the disinfectant . Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine . These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions . Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid. Y. Venkata Nancharaiah, 2003.We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation . A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating . Green and red fluorescent proteins were coexpressed in donor P . putida cells . Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone . Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy . Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation . This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments . To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies .
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