Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGF␣R) is fused to the cytosolic domain of Drosophila Torso (␣ Tor ) or the mouse fibroblast growth factor receptor 1 (␣ FR ). ␣ Tor homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGF␣R-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of ␣ Tor to stimulate the mitogen-activated protein (MAP) kinase pathway to near wildtype levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The ␣ FR chimeric receptor fails to rescue any aspect of the PDGF␣R-null phenotype. Instead, ␣ FR expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The ␣ FR phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.Evolutionary conservation of a gene requires that it contributes specific functions that enhance the fitness of the organism in which it is expressed. Receptor tyrosine kinases (RTKs) represent a large family of genes that have been conserved due to their ability to control multiple fundamental cellular processes. Upon binding to specific extracellular ligands, activated RTKs regulate cell proliferation, survival, migration, differentiation, and metabolism. The conservation of over 50 RTKs in mammals suggests that each executes critical specific cellular functions. How functional specificity is generated remains a matter of controversy. A vast amount of research has focused on understanding the molecular mechanisms that underlie the ability of these receptors to mediate diverse cellular functions. Surprisingly, studies have shown that even divergent RTKs activate highly redundant array of downstream signaling proteins, such as mitogen-activated protein (MAP) kinases, Src family kinases, phospholipase C (PLC)-␥, and phosphatidylinositol (PI) 3-kinase. Several models have been proposed to explain how RTKs achieve functional specificity at the cellular level while at the molecular level appearing to activate redundant signaling pathways (12,27).One model proposes that specific responses to RTKs are a result of differential activation of downstream pathways or biochemical differences in RTK signaling. A great deal of evidence based on experiments performed in cultured cell lines to assess individual contributions of downstream signaling pathways supports this idea (14,26). Differences in the strength and/or duration ...

A central issue in signal transduction is the physiological contribution of different growth factor-initiated signaling pathways. We have generated knockin mice harboring mutations in the PDGFalpha receptor (PDGFalphaR) that selectively eliminate its capacity to activate PI3 kinase (alpha(PI3K)) or Src family kinases (alpha(Src)). The alpha(PI3K) mutation leads to neonatal lethality due to impaired signaling in many cell types, but the alpha(Src) mutation only affects oligodendrocyte development. A third knockin line containing mutations that eliminate multiple docking sites does not increase the severity of the alpha(PI3K) mutation. However, embryos with mutations in the PI3K binding sites of both PDGFRs (alpha and beta) recapitulate the PDGFalphaR null phenotype. Our results indicate that PI3K has a predominant role in PDGFalphaR signaling in vivo and that RTK-activated signaling pathways execute both specific and overlapping functions during mammalian development.

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Background: The role of non-medical prescribers working in palliative care has been expanding in recent years and prescribers report improvements in patient care, patient safety, better use of health professionals' skills and more flexible team working. Despite this, there is a lack of empirical evidence to demonstrate its clinical and economic impact, limiting our understanding of the future role of non-medical prescribers within a healthcare system serving an increasing number of people with palliative care needs. Aim: We developed a unique methodology to establish the level of non-medical prescribers' activity in palliative care across England and consider the likely overall contribution these prescribers are making at a national level in this context in relation to medical prescribing. Setting/participants: All prescriptions for 10 core palliative care drugs prescribed by general practitioners, nurses and pharmacists in England and dispensed in the community between April 2011 and April 2015 were extracted from the Prescribing Analysis Cost Tool system. Design: The data were broken down by prescriber and basic descriptive analysis of prescription frequencies by opioid, non-opioids and total prescriptions by year were undertaken. To evaluate the yearly growth of non-medical prescribers, the total number of prescriptions was compared by year for each prescribing group. Results: Non-medical prescribers issued prescriptions rose by 28% per year compared to 9% in those issued by medical prescribers. Despite this, the annual growth in non-medical prescribers prescriptions was less than 1% a year in relation to total community palliative care prescribing activity in England. Impact on medical prescribing is therefore minimal.

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

Angiogenesis is a feature of over 50 different disease states. These include cancer, rheumatoid arthritis, cardiovascular diseases, diabetes and psoriasis. Methods developed to study these diseases are important tool to establish a model for testing potential therapeutics. These methods include both in vivo and in vitro assays. In vivo angiogenesis assays are considered to be the most informative because of the complex nature of vascular responses to test reagents and responses that no in vitro model can achieve. However in vivo assays can be expensive, laborious and require extensive training to perform. In contrast, in vitro assays can be carried out expeditiously, less costly and easier to interpret. Often, these in vitro assays provide maximum benefit when developed as a multivariate index assay where the data of multiple assays function to yield a composite result to interpret experimental studies. Using the Luminex xMAP technology, we have developed a multiplex Bio-Plex Pro™ Human Cancer Biomarker Panel I that employs a magnetic bead-based workflow to measure angiogenesis biomarkers in diverse matrices including serum, plasma, cell culture supernatant and many other sample types. In combination with the Bio-Plex® suspension array and the Bio-Plex Pro™ wash station, the multiplexing feature makes it possible to quantify the level of multiple angiogenesis targets in a single well of a 96-well microplate in just 3 hours, using as little as 12.5 μL of serum or plasma. The panel of 16 markers includes sEGFR, FGF basic, Follistatin, G-CSF, sHER2/neu, HGF, sIL-6Ra, Osteopontin, Leptin, PDGF-BB, PECAM-1, Prolactin, SCF, sTie-2, sVEGFR-1 and sVEGFR-2. These markers were selected because of their direct relevance to the pathogenesis of tumor-associated angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C6.

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