BackgroundSpiders (Order Araneae) are essential predators in every terrestrial ecosystem largely because they have evolved potent arsenals of silk and venom. Spider silks are high performance materials made almost entirely of proteins, and thus represent an ideal system for investigating genome level evolution of novel protein functions. However, genomic level resources remain limited for spiders.ResultsWe de novo assembled a transcriptome for the Western black widow (Latrodectus hesperus) from deeply sequenced cDNAs of three tissue types. Our multi-tissue assembly contained ~100,000 unique transcripts, of which > 27,000 were annotated by homology. Comparing transcript abundance among the different tissues, we identified 647 silk gland-specific transcripts, including the few known silk fiber components (e.g. six spider fibroins, spidroins). Silk gland specific transcripts are enriched compared to the entire transcriptome in several functions, including protein degradation, inhibition of protein degradation, and oxidation-reduction. Phylogenetic analyses of 37 gene families containing silk gland specific transcripts demonstrated novel gene expansions within silk glands, and multiple co-options of silk specific expression from paralogs expressed in other tissues.ConclusionsWe propose a transcriptional program for the silk glands that involves regulating gland specific synthesis of silk fiber and glue components followed by protecting and processing these components into functional fibers and glues. Our black widow silk gland gene repertoire provides extensive expansion of resources for biomimetic applications of silk in industry and medicine. Furthermore, our multi-tissue transcriptome facilitates evolutionary analysis of arachnid genomes and adaptive protein systems.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-365) contains supplementary material, which is available to authorized users.

Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption.We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E 2 (PGE 2 ), as an indicator of COX-2 activity, was measured using an enzyme immunoassay.Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68 + macrophages.

Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.

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Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption. We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E 2 (PGE 2), as an indicator of COX-2 activity, was measured using an enzyme immunoassay. Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68 + macrophages.

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.

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