Pathogenicity for Chickens of a Paramyxovirus Type 1, Isolated from Racing Pigeons in Sweden

Strains of paramyxovirus type 1 (PMV-1) have been isolated from diseased racing pigeons in Sweden. One of these isolates was selected for studies of the pathogenicity and contagiousness in chickens. The same isolate was previously found to have a high intravenous pathogenicity index (IVPI) in 6 weeks old chickens. In three experiments it was found that the PMV-1 isolate was very pathogenic for 1 week old chickens but not pathogenic for 120 day old pullets inoculated intranasally and ocularly. Symptoms in the young chickens were similar to those seen in the neurotropic form of Newcastle disease. The mortality was high and the incubation period 5–11 days. The disease easily spread to young chickens kept in contact with diseased birds. The microscopic examination revealed an interstitial nonpurulent pneumonia and a nonpurulent encephalitis in the young chickens. In the pullets the only finding was a mild encephalitis. PMV-1 was recovered from all young chickens but not from the pullets. Both the chickens and the inoculated pullets developed antibodies to PMV-1.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

The quantity of protein-bound [32P]phosphotyrosine in hepatocytes and fibroblasts. The effects of tyrosine protein kinase activating agents

Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.     

Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.     

Purification of synthetic peptides. Immobilized metal ion affinity chromatography (IMAC).

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.