A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Background

The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana.

Methods

Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba- 175 F- and C-segments by nested polymerase chain reaction.

Results

In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome.

Conclusion

The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.     

Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells

Immunostimulatory DNA sequences (ISS) containing CpG motifs induce interferon-α (IFN-α) and interferon-γ (IFN-γ) from human peripheral blood mononuclear cells and stimulate human B cells to proliferate and produce IL-6. We studied the motif and structural requirements for both types of activity using novel chimeric immunomodulatory compounds (CICs), which contain multiple heptameric ISS connected by non-nucleoside spacers in both linear and branched configurations. We found that the optimal motifs and structure for IFN-α production versus B cell activation differed. IFN-α production was optimal for CICs containing the sequences 5′-TCGXCGX and 5′-TCGXTCG, where X is any nucleotide. The presentation of multiple copies of these heptameric ISS with free 5′-ends via long, hydrophilic spacers, such as hexaethylene glycol, significantly enhanced the induction of IFN-α. Conversely, human B cell activity was predominately dependent on ISS motif, with 5′-TCGTXXX and 5′-AACGTTC being the most active sequences. Thus, we found CICs could be ‘programmed’ for IFN-α production or B cell activation as independent variables. Additionally, CICs with separate human- and mouse-specific motifs were synthesized and these were used to confirm in vivo activity in mice. CICs may offer unique advantages over conventional ISS because identification of the optimal motifs, spacers and structures for different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications.     

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.     

Phylogeographic structure and cryptic speciation in the trans-Antarctic moss Pyrrhobryum mnioides

Abstract Many bryophyte species have distributions that span multiple continents. The hypotheses historically advanced to explain such distributions rely on either long-distance spore dispersal or slow rates of morphological evolution following ancient continental vicariance events. We use phylogenetic analyses of DNA sequence variation at three chloroplast loci ( atpB-rbcL spacer, rps4 gene, and trnL intron and 3'spacer) to examine these two hypotheses in the trans-Antarctic moss Pyrrhobryum mnioides. We find: (1) reciprocal monophyly of Australasian and South American populations, indicating a lack of intercontinental dispersal; (2) shared haplotypes between Australia and New Zealand, suggesting recent or ongoing migration across the Tasman Sea; and (3) reciprocal monophyly among Patagonian and neotropical populations, suggesting no recent migration along the Andes. These results corroborate experimental work suggesting that spore features may be critical determinants of species range. We use the mid-Miocene development of the Atacama Desert, 14 million years ago, to calibrate a molecular clock for the tree. The age of the trans-Antarctic disjunction is estimated to be 80 million years ago, consistent with Gondwanan vicariance, making it among the most ancient documented cases of cryptic speciation. These data are in accord with niche conservatism, but whether the morphological stasis is a product of stabilizing selection or phylogenetic constraint is unknown.     

Surface contraction waves (SCWs) in the Xenopus egg are required for the localization of the germ plasm and are dependent upon maternal stores of the kinesin-like protein Xklp1

During the first four cell cycles in Xenopus, islands of germ plasm, initially distributed throughout the vegetal half of the egg cortex, move to the vegetal pole of the egg, fusing with each other as they do so, and form four large cytoplasmic masses. These are inherited by the vegetal cells that will enter the germ line. It has previously been shown that germ plasm islands are embedded in a cortical network of microtubules and that the microtubule motor protein Xklp1 is required for their localization to the vegetal pole [Robb, D., Heasman, J., Raats, J., and Wylie, C. (1996). Cell 87, 823-831]. Here, we show that germ plasm islands fail to localize and fuse in Xklp1-depleted eggs due to the abrogation of the global cytoplasmic movements known as surface contraction waves (SCWs). Thus, SCWs are shown to require a microtubule-based transport system for which Xklp1 is absolutely required, and the SCWs themselves represent a cortical transport system in the egg required for the correct distribution of at least one cytoplasmic determinant of future pattern.     

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Activation of kinetically distinct synaptic conductances on inhibitory interneurons by electrotonically overlapping afferents

We measured brain activity during mental rotation and object recognition with objects rotated around three different axes. Activity in the superior parietal lobe (SPL) increased proportionally to viewpoint disparity during mental rotation, but not during object recognition. In contrast, the fusiform gyrus was preferentially recruited in a viewpoint-dependent manner in recognition as compared to mental rotation. In addition, independent of the effect of viewpoint, object recognition was associated with ventral areas and mental rotation with dorsal areas. These results indicate that the similar behavioral effects of viewpoint obtained in these two tasks are based on different neural substrates. Such findings call into question the hypothesis that mental rotation is used to compensate for changes in viewpoint during object recognition.