transAlign: using amino acids to facilitate the multiple alignment of protein-coding DNA sequences

Background  

Alignments of homologous DNA sequences are crucial for comparative genomics and phylogenetic analysis. However, multiple alignment represents a computationally difficult problem. For protein-coding DNA sequences, it is more advantageous in terms of both speed and accuracy to align the amino-acid sequences specified by the DNA sequences rather than the DNA sequences themselves. Many implementations making use of this concept of "translated alignments" are incomplete in the sense that they require the user to manually translate the DNA sequences and to perform the amino-acid alignment. As such, they are not well suited to large-scale automated alignments of large and/or numerous DNA data sets.     

A regulated interaction between alpha5beta1 integrin and osteopontin

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.     

Dihydrozeatin metabolism in radish seedlings

(±)-[8-¹⁴C]Dihydrozeatin was fed to derooted radish seedlings. After three days the plants were harvested and the cytokinin metabolites purified     

Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase

Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)₂-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes.     

A Highlights from MBoC Selection: Identification and characterization of multiple novel Rab–myosin Va interactions

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.     

The hereditary hemochromatosis protein HFE and its chaperone beta2-microglobulin localise predominantly to the endosomal-recycling compartment

Hereditary Hemochromatosis is an iron overload disease most frequently associated with mutations in the HFE gene. While clinical studies of the disease have received extensive attention by various groups, the localisation, trafficking and function of the HFE protein, and its chaperone beta2-microglobulin (beta2M), require further investigation. In this study, we present data on the cellular localisation of HFE and its clinically relevant mutants in HuTu 80 cells. We find by confocal microscopy that HFE localises to the endosomal-recycling compartment (ERC), with minimal localisation to sorting or late endosomes. Interestingly, we also demonstrate that beta2M localises to the ERC where it co-localises with HFE. We find that exogenous expression of HFE results in enhanced beta2M cellular levels and that beta2M is necessary for cell surface expression of HFE. Finally, we have analysed the functional effects of exogenous expression of HFE and beta2M on transferrin binding to the cell surface. In summary, our study sheds light on the localisation and functional effects of the HFE and its chaperone protein beta2M.     

Ontogenetic Behavior and Migration of Atlantic Sturgeon, Acipenser oxyrinchus oxyrinchus, and Shortnose Sturgeon, A. brevirostrum, with Notes on Social Behavior

Ontogenetic behavior of Hudson River Atlantic sturgeon and Connecticut River shortnose sturgeon early life intervals were similar during laboratory observations. After hatching, free embryos were photonegative and sought cover. When embryos developed into larvae, fish left cover, were photopositive, and initiated downstream migration. Free embryos may remain at the spawning site instead of migrating downstream because the risk of predation at spawning sites is low. The two species are sympatric, but not closely related, so the similarities in innate behaviors suggest common adaptations, not phylogenetic relationship. Atlantic sturgeon migrated downstream for 12 days (peak, first 6 days), shortnose sturgeon migrated for 3 days, and year-0 juveniles of both species did not resume downstream migration. Short or long migrations of larvae may reflect different styles related to the total migratory distance from spawning sites to juvenile rearing areas. Atlantic sturgeon need to move a short distance to reach rearing areas and they had a long 1-step migration of 6–12 days. In contrast, shortnose sturgeon need to move a long distance to reach all rearing areas. This may be accomplished by a 2-step migration, of which the brief migration of larvae is only the first step. Early migrant Atlantic sturgeon were nocturnal, while late migrants were diurnal, and shortnose sturgeon were diurnal. These diel differences may also be adaptations for long (Atlantic sturgeon) or short (shortnose sturgeon) migrations. Cultured shortnose sturgeon, and possibly Atlantic sturgeon, have a dominance hierarchy with large fish dominant when competing for limited foraging space. Social behavior may be more important in the life history of wild sturgeons than is generally recognized.     

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.