Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

Crystallization of an endochitinase from Hordeum vulgar L. seeds.

Higher plants contain several constitutively expressed proteins for protection against infections by viruses, bacteria and fungi. Here we report the crystallization of a polypeptide with antifungal activity, a 26,000 dalton endochitinase from barley (Hordeum vulgare L.) seeds, in a form suitable for high-resolution X-ray analysis. Crystals were grown by vapor diffusion under several different conditions. The best crystals, obtained with ammonium sulfate as the precipitant, belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions a = b = 62.9 A and c = 96.0 A. The cell dimensions are consistent with one endochitinase molecule per asymmetric unit, and the crystals diffract to at least 2.0 A resolution.     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Domesticity of Lutzomyia whitmani (Diptera: psychodidae) populations: field experiments indicate behavioural differences

The sandfly Lutzomyia whitmani (Antunes & Coutinho) is an important vector for cutaneous leishmaniasis throughout much of Brazil, and has recently been shown to consist of more than one mitochondrial lineage. It has frequently been asserted that the degree of adaptation of L. whitmani to human environments varies across its range. As a standardized test of indoor feeding for three geographically distant populations of L. whitmani, catches inside experimental chicken sheds of varying degrees of wall closure (0%, 33%, 67% and 98%) were compared. Each increment in shed closure reduced catches of females (relative to the most open shed) by a similar degree for each population: geometric mean catches dropped by 11-40% with 33% closure, by 41-62% with 67% closure, and by 69-100% with 98% closure. Geometric mean catches of males from the two more northerly populations also decreased with increasing shed closure, by 18% and 22% for 33% closure, 58% and 69% for 67% closure, 91% and 93% for 98% closure. Males from the most southerly population showed significantly different behaviour, with 33% closure causing a 54% increase in geometric mean catch, 67% closure causing a 6% increase, and 98% closure causing a 32% reduction. For this southerly population, sex ratios became more male biased with increasing density in more closed sheds, suggesting aggregation driven by intra-specific communication. Lutzomyia intermedia (Lutz & Neiva) was relatively more likely than L. whitmani to approach baits in the three more closed sheds, rather than the most open shed, offering a behavioural explanation for observed differences in indoor biting rates between the species.     

Ca2+-dependent metarhodopsin inactivation mediated by calmodulin and NINAC myosin III

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination. M( *) was inactivated rapidly (tau approximately 20 ms) under control conditions, but approximately 10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M( *) inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M( *). Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.     

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.     

Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis

Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.