Risk of Infection with Leishmania spp. in the Canine Population in the Netherlands

The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania -endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey.     

Small-Angle x-ray scattering and crystallographic studies of arcelin-1: An insecticidal lectin-like glycoprotein from Phaseolus vulgaris L

Arcelin-1 and α-amylase inhibitor are two lectin-like glycoproteins expressed in the seeds of the kidney bean (Phaseolus vulgaris). They display insecticidal activities and protect the seeds from predation by larvae of various bruchids through different biological actions. Solution-state investigations by small-angle X-ray scattering (SAXS) show the dimeric structure of arcelin-1, a requirement for its hemagglutinating properties. Anions were found to have specific properties in their effectiveness to disrupt protein aggregates, affect solubility, and improve crystallizability. The SAXS results were used to improve crystallization conditions, and single crystals diffracting beyond 1.9 Å resolution were obtained. X-ray diffraction data analysis shows that noncrystallographic symmetry-related arcelin-1 molecules form a lectin-like dimer and reveals the presence of a solvent-exposed anion binding site on the protein, at a crystal-packing interface. The solution state properties of arcelin-1 and crystal twinning may be explained by the anion specificity of this binding site. Proteins 29:433–442, 1997. © 1997 Wiley-Liss, Inc.     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.     

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.     

Structural transitions in the FixJ receiver domain

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.