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Sulfadoxine Resistance in Plasmodium vivax Is Associated with a Specific Amino Acid in Dihydropteroate Synthase at the Putative Sulfadoxine-Binding Site. Michael Korsinczky, 2004.Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum . This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component . To investigate this mechanism of resistance by P . vivax to sulfadoxine, we cloned and sequenced the P . vivax dhps (pvdhps) gene . The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P . vivax as well as P . falciparum were created . The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species . The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P . vivax, equivalent to A613 in P . falciparum . V585 in P . vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P . vivax compared to P . falciparum . Sequencing dhps genes from a limited set of geographically different P . vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P . vivax to sulfadoxine . Additionally, amino acid mutations were observed in some P . vivax isolates in positions known to cause resistance in P . falciparum, suggesting that, as in P . falciparum, these mutations are responsible for acquired increases in resistance of P . vivax to sulfadoxine . Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type B. Maria Lövenklev, 2004.The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium . Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production . Multiple linear regression was used to set up six statistical models to quantify and predict these responses . All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate . In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method . This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay . CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases . The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere . These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml–1 · unit of optical density–1 in the 10% CO2 atmosphere and 126 ng · ml–1 · unit of optical density–1 in the 70% CO2 atmosphere . The Bacillus subtilis Signaling Protein SpoIVB Defines a New Family of Serine Peptidases. Ngo T. Hoa, 2002.The protein SpoIVB plays a key role in signaling in the  K checkpoint of Bacillus subtilis . This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase . SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-K to its active form,
K, in the outer mother cell chamber of the developing cell . In this work we have characterized the serine peptidase domain of SpoIVB . Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain . We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein . His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad . In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the
K checkpoint . The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily .
Molecular Characterization of Microsporidia Indicates that Wild Mammals Harbor Host-Adapted Enterocytozoon spp . as well as Human-Pathogenic Enterocytozoon bieneusi. Irshad M. Sulaiman, 2003.Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia . A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences . Fifty-nine PCR-positive samples were sequenced . Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp . (WL1 to WL17); of these, 15 have not been reported before . Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals . Some of the isolates from muskrats and raccoons formed two distinct groups . Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E . bieneusi . However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E . bieneusi and have no apparent public health significance . This is the first report of E . bieneusi in wildlife .
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