A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system.

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.     

Parasitism and the expression of sexual dimorphism

Although a negative covariance between parasite load and sexually selected trait expression is a requirement of few sexual selection models, such a covariance may be a general result of life‐history allocation trade‐offs. If both allocation to sexually selected traits and to somatic maintenance (immunocompetence) are condition dependent, then in populations where individuals vary in condition, a positive covariance between trait expression and immunocompetence, and thus a negative covariance between trait and parasite load, is expected. We test the prediction that parasite load is generally related to the expression of sexual dimorphism across two breeding seasons in a wild salamander population and show that males have higher trematode parasite loads for their body size than females and that a key sexually selected trait covaries negatively with parasite load in males. We found evidence of a weaker negative relationship between the analogous female trait and parasite infection. These results underscore that parasite infection may covary with expression of sexually selected traits, both within and among species, regardless of the model of sexual selection, and also suggest that the evolution of condition dependence in males may affect the evolution of female trait expression.     

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin–neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario—the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are ‘missing links’ in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).     

Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [β-³²P]UDP-glucose to [³²P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg²⁺ and was greatest at alkaline pH above 8. Kinetic analysis indicated a K_m of ∼4 mM for UDP-glucose and ∼0.3 mM for ADP-ribose. Based on V_max/K_m values, the enzyme was ∼20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M_r 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.     

Enteroaggregative strains of Escherichia coli belonging to serotypes 0126:H27 and 044:H18 express antigenically similar 18 kDa outer membrane-associated proteins

Abstract Outer membrane-associated proteins of 18 kDa were expressed by enteroaggregative Escherichia coli (EAggEC) belonging to serotypes 0126:H27 and O44:H18, which hybridized with a probe derived from a plasmid necessary for enteroaggregative adhesion. The 18 kDa proteins expressed by strains of E. coli , belonging to these serotypes, were surface exposed and antigenically similar but not structurally identical.     

Investigating the Structural Impact of the Glutamine Repeat in Huntingtin Assembly

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington’s disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ₄₂P₁₀) and compared the results with those obtained for the wild-type (NtQ₂₂P₁₀). Our results show that the aggregation pathway of NtQ₂₂P₁₀ is very different from that of NtQ₄₂P₁₀, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ₄₂P₁₀ are monomer and dimer. The divergent pathways ultimately result in NtQ₂₂P₁₀ fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ₄₂P₁₀ fibrils present a better fit to the Perutz β-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.     

Gallium-67 as a Potential Marker for Aluminium Transport in Rat Brain: Implications for Alzheimer''s Disease

Evidence of a link between aluminium and Alzheimer's disease, parkinsonism-dementia of Guam, and dialysis encephalopathy raises questions regarding the role of this element in the pathogenesis of these conditions. Therefore, we have investigated the use of gallium-67 (67Ga) as a marker for brain uptake of aluminium. The binding of 67Ga to plasma proteins has been studied, and the blood-brain barrier permeability and autoradiographic distribution of this isotope in rat brain determined in vivo. The autoradiographic distribution of 125I-Fe-transferrin receptors in rat brain has also been determined in vitro. Results show that 67Ga was bound to plasma transferrin, entered the brain with a blood-brain barrier permeability of 2.48 x 10(-6) ml/min/g, and showed a marked regional distribution that was very similar to that of 125I-Fe-transferrin receptors. Our data suggest that the vulnerability of the hippocampus, amygdala, and cerebral cortex in conditions such as those mentioned above may be partly due to an increased uptake and deposition of aluminium in these regions by the iron transport system.