Heme binding properties of heterologously expressed spinach cytochrome b(6): implications for transmembrane b-type cytochrome formation

In vivo and in vitro requirements for the formation of cytochrome b(6) were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b(6), formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b(6) appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b(6) was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.     

A primary assembly of a bovine haplotype block map based on a 15,036-single-nucleotide polymorphism panel genotyped in holstein-friesian cattle

Analysis of data on 1000 Holstein-Friesian bulls genotyped for 15,036 single-nucleotide polymorphisms (SNPs) has enabled genomewide identification of haplotype blocks and tag SNPs. A final subset of 9195 SNPs in Hardy-Weinberg equilibrium and mapped on autosomes on the bovine sequence assembly (release Btau 3.1) was used in this study. The average intermarker spacing was 251.8 kb. The average minor allele frequency (MAF) was 0.29 (0.05-0.5). Following recent precedents in human HapMap studies, a haplotype block was defined where 95% of combinations of SNPs within a region are in very high linkage disequilibrium. A total of 727 haplotype blocks consisting of > or =3 SNPs were identified. The average block length was 69.7 +/- 7.7 kb, which is approximately 5-10 times larger than in humans. These blocks comprised a total of 2964 SNPs and covered 50,638 kb of the sequence map, which constitutes 2.18% of the length of all autosomes. A set of tag SNPs, which will be useful for further fine-mapping studies, has been identified. Overall, the results suggest that as many as 75,000-100,000 tag SNPs would be needed to track all important haplotype blocks in the bovine genome. This would require approximately 250,000 SNPs in the discovery phase.     

Role of the Spatzle Pro-domain in the generation of an active toll receptor ligand

The cytokine Sp?tzle is the ligand for Drosophila Toll, the prototype of an important family of membrane receptors that function in embryonic patterning and innate immunity. A dimeric precursor of Sp?tzle is processed by an endoprotease to produce a form (C-106) that cross-links Toll receptor ectodomains and establishes signaling. Here we show that before processing the pro-domain of Sp?tzle is required for correct biosynthesis and secretion. We mapped two loss-of-function mutations of Sp?tzle to a discrete site in the pro-domain and showed that the phenotype arises because of a defect in biosynthesis rather than signaling. We also report that the pro-domain and C-106 remain associated after cleavage and that this processed complex signals with the same characteristics as the C-terminal fragment. These results suggest that before activation the determinants on C-106 that bind specifically to Toll are sequestered by the pro-domain and that proteolytic processing causes conformational rearrangements that expose these determinants and enables binding to Toll. Furthermore, we show that the pro-domain is released when the Toll extracellular domain binds to the complex, a finding that has implications for the generation of a signaling-competent Toll dimer.     

Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF(165). Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF(165) binding to NRP1, surprisingly reduces VEGF(121)-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF(121) binds directly to NRP1; however, unlike VEGF(165), VEGF(121) is not sufficient to bridge the NRP1.VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF(165), but not VEGF(121) binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.     

Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.     

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.     

Rapid intraoperative insulin assay: a novel method to differentiate insulinoma from nesidioblastosis in the pediatric patient

Background

The essential objectives for thyroidectomy are: avoidance of injury to the recurrent laryngeal nerves, conservation of the parathyroid glands, an accurate haemostasis and an excellent cosmesis. In the last 10 years major improvements and new technologies have been proposed and applied in thyroid surgery; among these mini-invasive thyroidectomy, regional anaesthesia and intraoperative neuromonitoring, and new devices for achieving dissection and haemostasis. Minor bleeding from small vessels could be a major complication in thyroid surgery. The purpose of ligating vessels is to maintain the surgical site free from an excess of blood and reduce blood loss in the patient.

Materials and methods

Hydroxylated polyvinyl acetal tampons (HPA) are made by a synthetic, open cell foam structure able to absorb fluids up to 25 times the initial weight. We tested their efficacy for small bleeding control and tissue dissection during several thyroid procedures.

Results

HPA tampons have been found extremely useful to absorb blood coming from minor and diffuse loss, helping to control bleeding by a combined action of fluid absorption and local compression. The porous design of the tampon allows the use of the suction device right through the tampon itself. Thanks to the initial mildly hard consistency, we also used HPA tampons as dissecting instruments.

Conclusion

In our experience the use of HPA tampons resulted extremely efficient for minor bleeding control, fluids removal and tissue dissection during thyroid surgery.     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.