Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.     

Impacts of Encouraging Dog Walking on Returns of Newly Adopted Dogs to a Shelter

This study involved examining the ability of a postadoption intervention to reduce returns of newly adopted dogs to shelters by encouraging physical activity between adopters and their dogs. Guardians in the intervention group received emails with dog behavior and human activity advice as well as invitations to join weekly dog walks. Both the intervention and control groups completed surveys regarding outdoor activity with their dogs, their dog-walking habits, and perceptions of their dogs’ behaviors. Adopter–dog pairs in the intervention group were not significantly more active than those in the control group, nor did they show a reduced incidence of returning their dogs. Guardians in both groups who reported higher obligation and self-efficacy in their dog walking were more active regardless of experimental condition; however, obligation, dog-walking self-efficacy, and perceptions about their dogs’ on-leash behaviors did not predict rates of return to the shelter. These findings add to the understanding of shelter dog re-relinquishment and the effective utilization of resources postadoption, and they indicate further research is needed to address the complexities of this newly forming human–dog relationship.     

Variable Selection and the Coexistence of Multiple mimetic forms of the Butterfly Heliconius numata

Polymorphism in aposematic animals and coexistence of multiple mimicry rings within a habitat are not predicted by classical Müllerian mimicry. The butterfly Heliconius numata Cramer (Lepidoptera: Nymphalidae; Heliconiinae) is both polymorphic and aposematic. The polymorphism is due to variation at a single locus (or `supergene') which determines colour patterns involved in Müllerian mimicry. We sampled 11 sites in a small area (approx. 60×30km) of North-eastern Peru for H. numata and its co-mimics in the genus Melinaea and Athyrtis (Ithomiinae), and examined the role of temporal and spatial heterogeneity in the maintenance of polymorphism. Colour-patterns of Melinaea communities, which constitute the likely `mimetic environment' for H. numata, are differentiated on a more local scale than morphs of H. numata, but the latter do show a strong and significant response to local selection for colour-pattern. In contrast, analysis of enzyme polymorphism in H. numata across the region revealed no spatial structure, which is consistent with a high mobility of this species. Differences in spatial variability in the two taxa may have caused H. numata to become polymorphic, while temporal variability, not significant in this study, probably has a lesser effect. The mimetic polymorphism is therefore explained by means of multiple selection-migration clines at a single locus, a similar process to that which explains narrow hybrid zones between geographic races of other Heliconius butterflies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Delineation of the Genera Struvea Bonder and Phyllodictyon J. E. Gray (Cladophorales, Chlorophyta)

Plants of the stalked, net-forming green alga Struvea plumosa Sender, the type species of the genus Struvea, divide segregatively at every stage of their multicellular differentiation. The segregative process results in virtually simultaneous internal cleavage of the cytoplasts of parent axes or laterals into uniseriate series of nearly identically sized daughter cells m which intercalary cross-wall formation never takes place. Several branch orders result through a repeated process by which each daughter cell produces a pair of opposite protrusions at its distal end; the protruded arms subsequently undergo segregative divisions themselves after reaching a sufficient length. Struvea elegans Børgesen is seemingly the only other member of the genus in which the thallus divides by this segregative process. The remaining species appear to lack segregative cell division, their septation resulting from non-synchronous, centripetal wall ingrowths that divide parent cells into more or less equal halves. Intercalary cell divisions are common, this process being easily seen in the most widely distributed member of the genus, Struvea anastomosans (Harv.) Pice, et Grunov ex Pice. Phyllodictyon J. E. Gray, based on Phyllodictyon putcherrimum. is currently considered a synonym of Struvea but should be reinstated to accommodate those former species of Struvea that have Cladophoratype. as opposed to segregative, cell division. Although the two genera thus differ substantially in their modes of cytokinesis and are assumed to represent independent developmental lines, both Struvea and Phyllodictyon are assigned to the Cladophorales on the basis of molecular studies by others showing that recognition of the separate order Siphonocladales renders the Cladophorales paraphyletic.     

Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.     

The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.