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.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.     

Species-specific effects of bombesin on gastric emptying and neurohypophyseal secretion

Previous reports have demonstrated that systemic injection of cholecystokinin (CCK) in rats produces dose-related decreases in food intake, increases in neurohypophyseal secretion of oxytocin (OT), and decreases in gastric emptying. The present studies determined whether systemic injection of bombesin (BBS), another peptide that potently reduces food intake in rats, had similar effects on OT secretion and gastric emptying. Although BBS produces a dose-dependent inhibition of food intake, even very high doses did not significantly affect plasma OT levels and only slightly decreased rates of gastric emptying. Consequently, despite their similar inhibitory effects on food intake, BBS does not appear to activate the same network of central nervous system pathways as does CCK in rats. However, parallel studies in monkeys demonstrated that systemic injection of BBS was effective in stimulating neurohypophyseal secretion of vasopressin rather than OT, in a pattern both qualitatively and quantitatively analogous to the effects of CCK in this species. Together with previous findings that BBS more potently inhibits gastric emptying in primates than in rats, these results therefore also suggest the presence of significant species differences in the central mechanisms by which BBS acts to reduce food intake.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats.

Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats.     

The ultrastructure and formation of the calcareous ossicles in the body wall of the sea cucumber Leptosynapta clarki (Echinodermata,Holothuroida)

Summary The calcareous ossicles of the burrowing sea cucumber Leptosynapta clarki have been examined by scanning and transmission electron microscopy. The ossicles occur in the dermis of the body wall and comprise three main types: 1) curved rods; 2) miliary granules; and 3) anchorshaped structures that are paired with oval plates. Rods average about 80 m in length, and miliary granules are typically 20–30 m long. Both of these ossicles appear to form a protective skeleton in regions where the water vascular system and accompanying nerves are located. Anchors and plates are scattered throughout the interambulacra of the body at densities ranging from 2–8/mm². Each anchor measures about 145 m long and is attached to the plate underlying it by a flexible ligament that is composed of collagen fibrils. Tetracycline labeling studies indicate that anchors and plates take several months to reach full size. All developing ossicles appear to be surrounded by a syncytial network of sclerocytes that characteristically possess numerous mitochondria and a conspicuous external lamina. Fully formed anchors lie directly beneath the epidermis and do not protrude through the outermost layer of the body wall. During burrowing, the curved flukes of the anchorshaped ossicles may provide added traction as the buccal tentacles dig through the sediment.List of abbreviations a anchor - ar ambulacral region - cm circular muscle layer of body wall - cs cytoplasmic sheath - d dermis - ep epidermis - f fluke - ild inner layer of dermis - k keel - me myoepithelium - mg miliary granule - n nerve - old outer layer of dermis - os ossicle - pl plate - s sclerocyte - sh shank - st stock     

Parkinson's disease: studies with an animal model

Parkinson' disease has been associated with degeneration of dopamine-containing neurons of the nigrostriatal bundle. Many neurological features of Parkinsonism can be produced in rats by selective destruction of central dopaminergic neurons using the neurotoxin 6-hydroxydopamine. In this review we discuss two aspects of Parkinson's disease that have been investigated in these animals. First, we consider why near-total degeneration of nigrostriatal bundle neurons is required before neurological symptoms emerge. It appears that the loss of dopaminergic neurons is accompanied by an exponential increase in the ratio of tyrosine hydroxylase activity to dopamine content. Thus, after the brain lesions there may be a compensatory increase in the capacity of residual dopaminergic neurons to synthesize and release transmitter. Second, we consider why stress produces severe neurological deficits in patients who are only mildly impaired otherwise. It appears that a variety of stressors produce an abrupt but transient increase in dopaminergic activity in the striatum of intact animals and that this increase is markedly attenuated by 6-hydroxydopamine treatment. Thus, stress-induced akinesia in animals with dopamine-depleting brain lesions and in Parkinsonian patients may result from the impaired ability of residual neurons to respond approximately to such stimuli.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Linkage Analysis with an Alternative Formulation for the Mixed Model of Inheritance: The Finite Polygenic Mixed Model

This paper presents an extension of the finite polygenic mixed model of FERNANDO et al. (1994) to linkage analysis. The finite polygenic mixed model, extended for linkage analysis, leads to a likelihood that can be calculated using efficient algorithms developed for oligogenic models. For comparison, linkage analysis of 5 simulated 4021-member pedigrees was performed using the usual mixed model of inheritance, approximated by HASSTEDT (1982), and the finite polygenic mixed model extended for linkage analysis presented here. Maximum likelihood estimates of the finite polygenic mixed model could be inferred to be closer to the simulated values in these pedigrees.