Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins ?1 and ?4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.     

A morphological,molecular, and histopathological redescription of Henneguya nyongensis Fomena & Bouix, 1996 (Cnidaria: Myxobolidae) infecting the gills of Peter’s elephantnose fish,Gnathonemus petersii (Günther) (Osteoglossiformes: Mormyridae), imported from Nigeria

Systematic Parasitology - A Henneguya sp., morphologically resembling Henneguya nyongensis Fomena & Bouix, 1996, was isolated from the gills of Peter’s elephantnose fish, Gnathonemus...     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

Applying proteomics to signaling networks

The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.     

Duodenal expression of the epithelial calcium transporter gene TRPV6: is there evidence for Vitamin D-dependence in humans?

Intestinal absorption of dietary calcium is regulated by 1,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) in humans and in experimental animals but interspecies differences in responsiveness to 1,25(OH)(2)D(3) are found, possibly due to differences in the promoters of genes for intestinal calcium transport proteins or of the Vitamin D receptor (VDR). The epithelial calcium transporter, known as ECAC2 or CAT1, the product of the TRPV6 gene expressed in proximal intestinal enterocytes, is the first step in calcium absorption and studies in mice have shown that its expression is Vitamin D-dependent. In contrast in man, we showed that duodenal TRPV6 mRNA expression was independent of blood 1,25(OH)(2)D(3), although in Caco-2 cells, 1,25(OH)(2)D(3)-dependent changes have been demonstrated. We sought to explain these findings. A consensus Vitamin D response element in the mouse gene is absent in the human gene. We re-analysed our duodenal expression data according to a CDX2-site polymorphism in the VDR promoter. Mean TRPV6 expression was the same, but there was evidence of different responsiveness to 1,25(OH)(2)D(3). In the GG genotype group, but not the AG, duodenal TRPV6 expression increased with 1,25(OH)(2)D(3). We postulate that lower levels of expression of VDR in the GG group produce greater sensitivity to 1,25(OH)(2)D(3).     

Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system

The aim of this study was to establish an in vitro system that supports development and differentiation of bovine blastocysts. Agar gel tunnels were covered with modified synthetic oviduct fluid medium supplemented with 10% fetal calf serum and 3g/l D-glucose. Of the total 67 blastocysts loaded individually into the tunnels, 46 continued expansion to 1mm and reached the walls of the gel on Day 10. On Day 12, 35 blastocysts elongated to minimum 1.6 mm while filling completely the space between the walls of the tunnel, and 16 still continued growth and reached an average of 4.3 mm length on Day 14. The largest blastocyst on Day 16 was 12 mm long. On Day 12, in 31 of the 35 elongated blastocysts a second cell layer occurred beneath the trophoblast and formed a complete cover in surviving Day 14 embryos. In most proliferating embryos the inner cell mass was prominent, however, the detection of signs of embryonic disc formation will require further studies. The established system was suitable to induce in vitro elongation, rapid growth and further differentiation, and may have considerable theoretical and practical value for studies of development and differentiation of bovine embryos.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.