Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.     

A novel class of achiral seco-analogs of CC-1065 and the duocarmycins: design, synthesis, DNA binding, and anticancer properties

The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylindoline (or achiral seco-CI) analogs (5a-o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a 4-hydroxyphenethyl halide moiety that is attached to a wide range of indole, benzimidazole, pyrrole, and pyridyl-containing noncovalent binding components. The 4-hydroxyphenethyl halide moiety represents the simplest mimic of the seco-cyclopropylpyrroloindoline (seco-CPI) pharmacophore found in the natural products, and it lacks a chiral center. The sequence and minor groove specificity of the achiral compounds was ascertained using a Taq DNA polymerase stop assay and a thermal induced DNA cleavage experiment using either a fragment of pBR322 or pUC18 plasmid DNA. For example, seco-CI-InBf (5a) and seco-CI-TMI (5c) demonstrated specificity for AT-rich sequences, particularly by reacting with the underlined adenine-N3 position of 5'-AAAAA(865)-3'. This is also the sequence that CC-1065 and adozelesin prefer to alkylate. The achiral seco-CI compounds were subjected to cytotoxicity studies against several human (K562, LS174T, PC3, and MCF-7) and murine cancer cell lines (L1210 and P815). Following continuous drug exposure, the achiral compounds were found to be cytotoxic, with IC(50) values in the muM range. Interestingly, the carbamate protected compound 5p was significantly less cytotoxic than agent 5c, supporting the hypothesis that loss of HCl and formation of a spiro[2,5]cyclopropylcyclohexadienone intermediate is necessary for biological activity. The achiral seco-CI compounds 5a and 5c were submitted to the National Cancer Institute for further cytotoxicity screening against a panel of 60 different human cancer cell lines. Both compounds showed significant activity, particularly against several solid tumor cell lines. Flow cytometry studies of P815 cells that were incubated with compound 5c at its IC(50) concentration for 24h showed induction of apoptosis in a large percentage of cells. Compounds 5a and 5c were selected by the NCI for an in vivo anticancer hollow-fiber test, and received composite scores of 18 and 22, respectively. These two compounds were subsequently evaluated for in vivo anticancer activity against the growth of a human advanced stage SC UACC-257 melanoma in skid mice. At a dose of 134 mg/kg administered IP, compound 5c gave a T/C value of 40% (for day 51), and the median number of days of doubling tumor growth was 27.7, versus 15.8 for untreated animals. For compound 5a, at 200mg/kg, the T/C was 58% and the median number of days of doubling tumor growth was 20.0 versus 8.7 for untreated animals. At these doses no toxicity or weight loss was observed for either compound. Furthermore, compound 5c was not toxic to murine bone marrow cell growth in culture, at a dose that was toxic for the previously reported seco-CBI (cyclopropylbenzoindoline)-TMI (4).     

Mechanisms of organophosphate insecticide-induced airway hyperreactivity

It has been suggested that pesticide exposure may be a contributing factor underlying the increased incidence of asthma in the United States and other industrialized nations. To test this hypothesis, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used organophosphate pesticide. Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 h or 7 days after a single subcutaneous injection of either 390 mg/kg or 70 mg/kg of chlorpyrifos, respectively. Mechanisms by which chlorpyrifos may cause airway hyperreactivity include inhibition of acetylcholinesterase (AChE) or dysfunction of M3 muscarinic receptors on airway smooth muscle or of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 h after treatment with 390 mg/kg of chlorpyrifos, but not 7 days after injection of 70 mg/kg of chlorpyrifos. Acute exposure to eserine (250 microg/ml) also significantly inhibited lung AChE but did not potentiate vagally induced bronchoconstriction. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifos-treated animals, pilocarpine dose-response curves were shifted significantly to the right, demonstrating decreased responsiveness of neuronal M2 receptors. In contrast, chlorpyrifos treatment did not alter methacholine-induced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway smooth muscle. These data demonstrate that organophosphate insecticides can cause airway hyperreactivity in the absence of AChE inhibition by decreasing neuronal M2 receptor function.     

Insight in schizophrenia spectrum disorders: relationship with behavior,mood and perceived quality of life,underlying causes and emerging treatments

Poor insight in schizophrenia is prevalent across cultures and phases of illness. In this review, we examine the recent research on the relationship of insight with behavior, mood and perceived quality of life, on its complex roots, and on the effects of existing and emerging treatments. This research indicates that poor insight predicts poorer treatment adherence and therapeutic alliance, higher symptom severity and more impaired community function, while good insight predicts a higher frequency of depression and demoralization, especially when coupled with stigma and social disadvantage. This research also suggests that poor insight may arise in response to biological, experiential, neuropsychological, social‐cognitive, metacognitive and socio‐political factors. Studies of the effects of existing and developing treatments indicate that they may influence insight. In the context of earlier research and historical models, these findings support an integrative model of poor insight. This model suggests that insight requires the integration of information about changes in internal states, external circumstances, others’ perspectives and life trajectory as well as the multifaceted consequences and causes of each of those changes. One implication is that treatments should, beyond providing education, seek to assist persons with schizophrenia to integrate the broad range of complex and potentially deeply painful experiences which are associated with mental illness into their own personally meaningful, coherent and adaptive picture.     

Ecosystem‐based management of coral reefs under climate change

Coral reefs provide food and livelihoods for hundreds of millions of people as well as harbour some of the highest regions of biodiversity in the ocean. However, overexploitation, land‐use change and other local anthropogenic threats to coral reefs have left many degraded. Additionally, coral reefs are faced with the dual emerging threats of ocean warming and acidification due to rising CO₂ emissions, with dire predictions that they will not survive the century. This review evaluates the impacts of climate change on coral reef organisms, communities and ecosystems, focusing on the interactions between climate change factors and local anthropogenic stressors. It then explores the shortcomings of existing management and the move towards ecosystem‐based management and resilience thinking, before highlighting the need for climate change‐ready marine protected areas (MPAs), reduction in local anthropogenic stressors, novel approaches such as human‐assisted evolution and the importance of sustainable socialecological systems. It concludes that designation of climate change‐ready MPAs, integrated with other management strategies involving stakeholders and participation at multiple scales such as marine spatial planning, will be required to maximise coral reef resilience under climate change. However, efforts to reduce carbon emissions are critical if the long‐term efficacy of local management actions is to be maintained and coral reefs are to survive.     

Extensive gypsy moth defoliation in Southern New England characterized using Landsat satellite observations

Southern New England is currently experiencing the first major gypsy moth (Lymantria dispar) defoliation event in nearly 30 years. Using a novel approach based on time series of Landsat satellite observations, we generated consistent maps of gypsy moth defoliation for 2015 (first year of the outbreak), 2016 (second year of outbreak), and 2017 (third year of outbreak). Our mapped results demonstrate that the defoliation event continued through the 2017 growing season. Moreover, the affected area more than doubled in extent each year and expanded radially to encompass 4386 km² of forested area in Rhode Island, eastern Connecticut, and central Massachusetts. The current gypsy moth outbreak is believed to be the result of a series of unusually dry springs in 2014, 2015, and 2016, which suppressed Entomophaga maimaiga, a fungal mortality agent that has historically reduced gypsy moth impacts in this region. The continuation and marked expansion of the outbreak in 2017 despite average spring rainfall suggests that caterpillars were active early in the growing season, and mortality from the fungus likely peaked after significant defoliation had already occurred. Our Landsat time series approach represents an important new source of data on spatial and temporal patterns in gypsy moth defoliation, and continued satellite-based monitoring will be essential for tracking the progress of this and other gypsy moth outbreaks.     

5-HT2 Receptors Facilitate JC Polyomavirus Entry

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT_2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT_2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT₂ receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT₂ receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT₂ receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.