c-Jun N-terminal kinase (JNK) cooperates with Gsk3β to regulate Dishevelled-mediated microtubule stability

Background  

Wnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3β regulates MT dynamics.     

Discs of mammalian rod photoreceptors form through the membrane evagination mechanism

Photoreceptor discs are membrane organelles harboring components of the visual signal transduction pathway. The mechanism by which discs form remains enigmatic and is the subject of a major controversy. Classical studies suggest that discs are formed as serial plasma membrane evaginations, whereas a recent alternative postulates that discs, at least in mammalian rods, are formed through intracellular vesicular fusion. We evaluated these models in mouse rods using methods that distinguish between the intracellular vesicular structures and plasma membrane folds independently of their appearance in electron micrographs. The first differentiated membranes exposed to the extracellular space from intracellular membranes; the second interrogated the orientation of protein molecules in new discs. Both approaches revealed that new discs are plasma membrane evaginations. We further demonstrated that vesiculation and plasma membrane enclosure at the site of new disc formation are artifacts of tissue fixation. These data indicate that all vertebrate photoreceptors use the evolutionary conserved membrane evagination mechanism to build their discs.     

First record of a spawning aggregation for the tropical eastern Pacific endemic grouper Mycteroperca olfax in the Galapagos Marine Reserve

This study provides direct and indirect evidence of temporally and spatially consistent spawning aggregations for the grouper Mycteroperca olfax. Recently reported declines in population numbers, probably related to the direct targeting of aggregations by artisanal fishermen, highlight the urgent need for species‐specific management actions in the Galapagos Marine Reserve, such as minimum and maximum landing sizes, and the importance of protecting key aggregation sites with the declaration of no‐take areas and the establishment of total fishing bans during the reproductive season.     

The search for stability on narrow supports: an experimental study in cats and dogs

Kinematic and coordination variables were studied in two carnivorans, one with known locomotor capabilities in arboreal substrates (cat), and the other a completely terrestrial species (dog). Two horizontal substrates were used: a flat trackway on the ground (overground locomotion) and an elevated and narrow runway (narrow-support locomotion). Despite their different degree of familiarity with the ‘arboreal’ situation, both species developed a strategy to adapt to narrow supports. The strategy of cats was based on using slower speeds, coupled with modifications to swing phase duration, to keep balance on narrow supports. The strategy of dogs relied on high speeds to gain in dynamic stability, and they increased cycle frequency by reducing swing phase duration. Furthermore, dogs showed a high variability in limb coordination, although a tendency to canter-like coordination was observed, and also avoided whole-body aerial phases. In different ways, both strategies suggested a reduction of peak vertical forces, and hence a reduction of the vertical oscillations of the centre of mass. Finally, lateral oscillation was reduced by the use of a crouched posture.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

Tuned Escherichia coli as a host for the expression of disulfide-rich proteins

Disulfide-bond formation is a major post-translational modification and is essential for protein folding, stability, and function. This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Focusing on the use of Escherichia coli for the production of this class of proteins, we describe the mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those that promote the formation and isomerization of disulfide bonds in the bacterial periplasm, while presenting parallel pathways in the eukaryotic endoplasmic reticulum. Based on these concepts, we review the use of E. coli as a cell factory for the production of heterologous disulfide-containing proteins using either peri- or cytoplasmic expression and, in particular, how these compartments can be tuned to improve the yield of correctly folded recombinant proteins. Finally, we describe a few examples of the production of small disulfide-rich proteins (protease inhibitors) to illustrate how soluble, active, and fully oxidized recombinants may be successfully obtained upon peri- or cytoplasmic expression in E. coli.     

Continuous-flow/stopped-flow system for enzyme immunoassay using a rotating bioreactor: determination of Chagas disease

The high sensitivity that can be attained using an immunoassays coupled to a rotating bioreactor with electrochemical detection mediated by [Os(bpy)2Cl(pyCOOH)]Cl, has been verified for the detection of Trypanozoma cruzi (T. cruzi), This protozoan parasite causes Chagas disease, affecting more than 18 million people in central and south America. Antibodies in the serum sample are allowed to react immunologically with whole homogenates of the parasite as antigen that are immobilized on a rotating disk. The bound antibodies are quantified by a horseradish peroxidase (HRP) enzyme labeled second antibodies specific to human IgG in presence of hydrogen peroxide using an osmium complex [Os(bpy)2Cl(pyCOOH)]Cl as enzymatic mediators. The amperometric measurement performed at 0.00 V versus Ag/AgCl can be done within 2 min and the analysis time does not exceed 23 min. The calculated detection limits was 0.01 mIU ml(-1). Reproducibility assays were made using repetitive serum of 0.182 mIU ml(-1) T. cruzi specific antibody (measured as the activity of the correspondent anti-serum's enzyme conjugated); the percentage standard error was less than 5%. The amperometric immunoreactors showed significantly higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method.     

Evolution of selenium utilization traits

Background

The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor.

Results

We analyzed completely sequenced genomes for occurrence of the selA, B, C, D and ybbB genes. We found that selB and selC are gene signatures for the Sec-decoding trait. However, selD is also present in organisms that do not utilize Sec, and shows association with either selA, B, C and/or ybbB. Thus, selD defines the overall selenium utilization. A global species map of Sec-decoding and 2-selenouridine synthesis traits is provided based on the presence/absence pattern of selenium-utilization genes. The phylogenies of these genes were inferred and compared to organismal phylogenies, which identified horizontal gene transfer (HGT) events involving both traits.

Conclusion

These results provide evidence for the ancient origin of these traits, their independent maintenance, and a highly dynamic evolutionary process that can be explained as the result of speciation, differential gene loss and HGT. The latter demonstrated that the loss of these traits is not irreversible as previously thought.     

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.