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.     

Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight.

The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.     

Disentangling invasion processes in a dynamic shipping-boating network

The relative importance of multiple vectors to the initial establishment, spread and population dynamics of invasive species remains poorly understood. This study used molecular methods to clarify the roles of commercial shipping and recreational boating in the invasion by the cosmopolitan tunicate, Botryllus schlosseri. We evaluated (i) single vs. multiple introduction scenarios, (ii) the relative importance of shipping and boating to primary introductions, (iii) the interaction between these vectors for spread (i.e. the presence of a shipping-boating network) and (iv) the role of boating in determining population similarity. Tunicates were sampled from 26 populations along the Nova Scotia, Canada, coast that were exposed to either shipping (i.e. ports) or boating (i.e. marinas) activities. A total of 874 individuals (c. 30 per population) from five ports and 21 marinas was collected and analysed using both mitochondrial cytochrome c oxidase subunit I gene (COI) and 10 nuclear microsatellite markers. The geographical location of multiple hotspot populations indicates that multiple invasions have occurred in Nova Scotia. A loss of genetic diversity from port to marina populations suggests a stronger influence of ships than recreational boats on primary coastal introductions. Population genetic similarity analysis reveals a dependence of marina populations on those that had been previously established in ports. Empirical data on marina connectivity because of boating better explains patterns in population similarities than does natural spread. We conclude that frequent primary introductions arise by ships and that secondary spread occurs gradually thereafter around individual ports, facilitated by recreational boating.     

Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.     

Effect of Inflammatory Stimuli on the Silver Staining Pattern of the Rat Carotid Endothelium

Silver nitrate stains the intercellular junctions of the endothelium and other cytoplasmic or membrane components. Two protocols are described for the silver staining of rat carotid endothelium that exclude the use of pressurized fixatives and simplify the technique previously described for rat aorta. The entire surface of the carotid endothelium was examined and several parameters (stigmata, granularity, clustering of anionic sites, transversal lines, weakening of silver lines and leukocyte adhesion) were evaluated. We studied the pattern of silver staining in two situations: (1) endothelial activation and (2) neurogenic inflammation. Endothelial activation was produced by the intravenous administration of a proinflammatory albumin or polyinosinic acid. Both products cause a marked increase in leukocyte adhesion concomitant with a decrease in argyrophilia and a weakness or loss of silver lines. Neurogenic inflammation, which is mediated by substances released from sensory nerves, was induced by the intravenous administration of substance P or capsaicin. Both stimuli produced an increase in argyrophilia and weakness or loss of silver lines. Substance P caused a clustering of anionic sites, whereas this phenomenon was more discrete with capsaicin. Nearly 80% of all examined rats (controls and inflammatory stimuli treated) showed endothelial membrane disruptions formed by clusters of cells often in the shape of streaks aligned with the long axis of the vessel. The detection of these discontinuities is important, as loss of endothelial integrity is central in the initiation of pathological events.     

Reassessment of the vocal repertoire of a nest-building gladiator frog,Boana pardalis (Anura,Hylidae, Cophomantinae): implications for its diagnosis within the B. faber species group

In this study, we re-describe the advertisement and territorial calls of Boana pardalis, carry out an acoustic comparison between the studied species and the other congeners of the B. faber group, and report for the first time the tympanic amplexus for the studied species. The advertisement call of B. pardalis can be used to supplement its diagnosis in the B. faber group based on temporal call traits, e.g. emission rate and emission pattern, as well as the call envelope.     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997