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.

Mass spectrometric analysis of rat cerebrospinal fluid proteins following exposure to the neurotoxicant carbonyl sulfide.

RATIONALE: Using a proteomic-based approach we have investigated possible altered expression of a range of cerebral spinal fluid (CSF) proteins following exposure to the neurotoxicant carbonyl sulfide (COS). CSF is ideal for the investigation of markers of brain injury or disease since it is secreted from several central nervous system structures and changes in the CSF composition may reflect brain insult and many pathological processes. METHODS: Animals were placed in exposure chambers and were exposed to 0 ppm or 500 ppm COS for 1, 2 or 3 days, 6 h per day. After the last inhalation exposure, 50-70 µL CSF sample was obtained by lumbar puncture. CSF samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) on either a Premier quadrupole time-of-flight (QTOF) or an Agilent 6340 ion trap and by matrix-assisted laser desorption/ionization (MALDI)-MS on a 4800 MALDI-TOF/TOF analyzer. RESULTS: The dynamic range of abundance of the identified proteins spanned over more than three orders of magnitude. The four most abundant proteins identified (albumin, cystatin C, serotransferrin, transthyretin) are major proteins that are present in both CSF and blood at high levels but the fifth most abundant protein identified (prostaglandin H2D isomerase) is the second most abundant protein in human CSF and is secreted and synthesized in the rat central nervous system. No significant differences were observed between COS-treated CSF samples and the control CSF samples because of blood contamination. CONCLUSIONS: Quantitative MS protein analyses of rat CSF is limited by the low sample volumes that can practicably be obtained from rats and the low protein concentrations in rat CSF. Results of this work suggest a clear need for CSF collection that would minimize blood contamination. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Sex determination in platypus and echidna: autosomal location of SOX3 confirms the absence of SRY from monotremes.

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.

Cloning and mapping of platypus SOX2 and SOX14: insights into SOX group B evolution.

Group B SOX genes, the closest relatives to the sex-determining gene SRY, are thought to have evolved from a single ancestral SOX B by a series of duplications and translocations. The two SOX B genes SOX2 and SOX14 co-localize to chromosome 3q in humans. SOX2 and SOX14 homologues were cloned and characterized in the platypus, a monotreme mammal distantly related to man. The two genes were found to co-localize to chromosome 1q in this species. Proximity of the two related genes has therefore been conserved for 170 Myr, since humans and platypus diverged. The sequence similarity and conserved synteny of these group B genes provide clues to their origin. A simple model of SOX group B gene evolution is proposed.

Nosocomial Escherichia coli O157 infection.

Nosocomial transmission of Shiga toxin-producing Escherichia coli O157 to two patients and three nurses is described. The index case presented with rectal bleeding rather than diarrhoea, and additional infection control measures were therefore only instituted after detection of the organism. Of the nurses, two were asymptomatic and detected on a screening programme of all staff in contact with the affected patients. Two patients died, one from Clostridium perfringens bacteraemia. The use by staff of full protective gowns for handling patients from their first onset of diarrhoea is recommended, rather than plastic aprons. Interest from the media was intense, despite the small number of patients and staff affected, and early preparations for media enquiries should be made in such episodes.

Relation between catecholamine-induced cyclic AMP changes and hyperpolarization in isolated rat sympathetic ganglia.

1. The effect of catecholamines on cyclic adenosine 3'5'-monophosphate (cyclic AMP) production in isolated rat superior cervical ganglia has been measured under experimental conditions in which they also produce ganglion hyperpolarization.2. (+/-)Isoprenaline (1 muM) increased cyclic AMP levels by 8-100 times after 15 min incubation at 25 degrees C. Half-maximal stimulation occurred at about 0.03 muM. This was due to stimulation of beta-receptors, since it was prevented by 1 muM-propranolol but not by 1 muM-phentolamine.3. The alpha-agonists phenylephrine (100 muM), dopamine (100 muM) and clonidine (1 muM) did not produce a detectable increase in ganglionic cyclic AMP. Dopamine (100 muM) was also ineffective at 37 degrees C in the presence of 10 mM-theophylline.4. Exogenous cyclic AMP (0.01-1 mM) hyperpolarized the ganglion. This effect was replicated by other adenosine compounds, most effectively by adenosine and by adenosine 5'-monophosphate, and was antagonized by theophylline. Dibutyryl cyclic AMP was weaker than cyclic AMP.5. Neither theophylline nor the non-xanthine phosphodiesterase inhibitor, Ro 20-1724, enhanced the hyperpolarizing actions of noradrenaline or dopamine.6. Since catecholamine-induced hyperpolarization of the isolated rat ganglion is induced via alpha-receptors, whereas cyclic AMP-production is induced via beta-receptors, it is concluded that cyclic AMP is unlikely to mediate the hyperpolarization. The effect of exogenous cyclic AMP may be due to an action on external adenosine-receptors.

Inhibition of human lymphocyte transformation as an in vitro parallel line bioassay for anti-human lymphocyte globulin.

Horse anti-human lymphocyte globulin (HALG) is now widely clinically, but the variable immunosuppressive potency of different preparations of HALG has necessitated development of an accurate, reproducible in vitro assay of HALG potency. Currently available tests have several disadvantages, as well as showing little correlation with in vivo activity of the preparations tested. Incorporation of tritiated thymidine into lymphocytes, stimulated with mitogen (PHA) or antigen (PPD) and the inhibition of this process by HALG is described. ID50S and potency ratios have been determined for four HALG preparations. The ID50S obtained with these preparations were reproducible and the potency ratios obtained using 3 + 3 parallel line bioassay were similarly reproducible, a change in rank order being observed only once in ten assays. These in vitro results correlate with in vivo skin graft data. It is suggested that this technique could be used for evaluation of HALG preparations on peripheral blood from potential recipients.

Defective PGE reactivity in leucocytes of multiple sclerosis patients.

Leucocyte migration inhibition in vitro, in response to antigen or mitogen, is suppressed by PGE2 (0.01-1.0 mug/ml). The susceptibility of leucocytes to such inhibition by PGE2 has been compared using cell preparations obtained from normal individuals, multiple sclerosis patients and from patients with other neurological diseases. The results indicate defective reactivity of leucocytes from multiple sclerosis patients.