The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF-α to activate apoptosis.

Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.

Financing and organisation of veterinary services.

This paper analyses the different ways of financing official Veterinary Services (VS) and the effects of these choices on the performance of such Services. The links between governance, organisational effectiveness and financing arrangements are seen as particularly important. The paper comments on some of the advantages and disadvantages of financing VS with service fees, as compared to budget transfers from general government revenues. Evidence is presented on the considerable heterogeneity in the size of VS and on the impact of this heterogeneity on organisation and financing. The paper concludes with a stylised case study, which emphasises the importance of collaboration and the division of labour between the official and the private sector of the veterinary profession.

Effect of proteasome inhibitors with different chemical structures on the ubiquitin-proteasome system in vitro.

Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.

Cation currents in human airway epithelial cells induced by infection with influenza A virus.

Influenza A viruses cause lung disease via an incompletely understood mechanism that involves the accumulation of liquid within the lungs. The accumulation of lung liquid is normally prevented by epithelial Na(+) absorption, a transport process regulated via several pathways including phosphoinositide-3-kinase (PI3K). Since the influenza A virus encodes a non-structural protein (NS1) that can activate this kinase, we now explore the effects of NS1 upon the biophysical properties of human airway epithelial cells. Transient expression of NS1 depolarized electrically isolated cells maintained in glucocorticoid-free medium by activating a cation conductance identical to the glucocorticoid-induced conductance seen in single cells. This response involved PI3K-independent and PI3K-dependent mechanisms. Infecting glucocorticoid-deprived cells with influenza A virus disrupted the normal electrical coupling between neighbouring cells, but also activated a conductance identical to that induced by NS1. This response to virus infection was only partially dependent upon NS1-mediated activation of PI3K. The presence of NS1 allows influenza A to modify the biophysical properties of infected cells by activating a Na(+)-permeable conductance. Whilst the activation of Na(+)-permeable channels may be expected to increase the rate of Na(+) absorption and thus reduce the volume of liquid in the lung, liquid does normally accumulate in influenza A-infected lungs. The overall effect of influenza A on lung liquid volume may therefore reflect a balance between the activation and inhibition of Na(+)-permeable channels.

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation.

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na(+) conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 muM) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na(+) absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Psychometric properties of the Observer Alexithymia Scale in a clinical sample.

The purpose of this study was to evaluate the psychometric properties of the Observer Alexithymia Scale (OAS; Haviland, Warren, & Riggs, 2000) in a clinical setting. Clinical and counseling psychologists used the OAS to rate outpatients (n = 192) with various Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 1994) diagnoses. Reliability and validity data are similar to the initial nonclinical data (n = 819): OAS scores are reliable (coefficient alpha = .90), and the five-factor structure--Distant, Uninsightful, Somatizing, Humorless, and Rigid--was confirmed. Moreover, the OAS does a relatively good job of differentiating clinical from nonclinical cases. The OAS is psychometrically sound, and it appears to be a useful tool for collecting and evaluating observer data on the clinically relevant, everyday expressions of alexithymia.

Effects of drugs and of electrical stimulation on the muscularis mucosae of rabbit large intestine.

1. The muscularis mucosae of the rabbit large intestine shows spontaneous rhythmic contractions with an average frequency of about 11/minute.2. The muscularis mucosae responds to acetylcholine, adrenaline and noradrenaline in a manner similar to that of whole segments of terminal large intestine.3. The muscularis mucosae would seem to have a cholinergic motor innervation.