The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands

Cytochromes P450 of the CYP2C and CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET omega-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-alpha showed that omega-hydroxylated 14,15-EET bound to this receptor with high affinity (K(i) = 3 +/- 1 nm). Moreover, at 1 microm, the omega-alcohol of 14,15-EET or a 1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-alpha in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.     

Selective substrate supply in the regulation of yeast de novo sphingolipid synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.     

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.     

Rigidified 2-aminopyrimidines as histamine H4 receptor antagonists: Effects of substitution about the rigidifying ring

Three novel series of histamine H₄ receptor (H₄R) antagonists containing the 2-aminopyrimidine motif are reported. The best of these compounds display good in vitro potency in both functional and binding assays. In addition, representative compounds are able to completely block itch responses when dosed ip in a mouse model of H₄-agonist induced scratching, thus demonstrating their activities as H₄R antagonists.     

Evaluation of a Commercial Latex Agglutination Test Kit for Cryptococcal Antigen

Two dozen Crypto-LA kits for detecting Cryptococcus neoformans capsular polysaccharide antigens were evaluated. Ten kits proved reliable for detecting and titering antigen in clinical materials. Fourteen kits were found to be inadequate.     

From Data towards Knowledge: Revealing the Architecture of Signaling Systems by Unifying Knowledge Mining and Data Mining of Systematic Perturbation Data

Genetic and pharmacological perturbation experiments, such as deleting a gene and monitoring gene expression responses, are powerful tools for studying cellular signal transduction pathways. However, it remains a challenge to automatically derive knowledge of a cellular signaling system at a conceptual level from systematic perturbation-response data. In this study, we explored a framework that unifies knowledge mining and data mining towards the goal. The framework consists of the following automated processes: 1) applying an ontology-driven knowledge mining approach to identify functional modules among the genes responding to a perturbation in order to reveal potential signals affected by the perturbation; 2) applying a graph-based data mining approach to search for perturbations that affect a common signal; and 3) revealing the architecture of a signaling system by organizing signaling units into a hierarchy based on their relationships. Applying this framework to a compendium of yeast perturbation-response data, we have successfully recovered many well-known signal transduction pathways; in addition, our analysis has led to many new hypotheses regarding the yeast signal transduction system; finally, our analysis automatically organized perturbed genes as a graph reflecting the architecture of the yeast signaling system. Importantly, this framework transformed molecular findings from a gene level to a conceptual level, which can be readily translated into computable knowledge in the form of rules regarding the yeast signaling system, such as “if genes involved in the MAPK signaling are perturbed, genes involved in pheromone responses will be differentially expressed.”     

First Record of Eocene Bony Fishes and Crocodyliforms from Canada’s Western Arctic

Background

Discovery of Eocene non-marine vertebrates, including crocodylians, turtles, bony fishes, and mammals in Canada’s High Arctic was a critical paleontological contribution of the last century because it indicated that this region of the Arctic had been mild, temperate, and ice-free during the early – middle Eocene (∼53–50 Ma), despite being well above the Arctic Circle. To date, these discoveries have been restricted to Canada’s easternmost Arctic – Ellesmere and Axel Heiberg Islands (Nunavut). Although temporally correlative strata crop out over 1,000 km west, on Canada’s westernmost Arctic Island – Banks Island, Northwest Territories – they have been interpreted as predominantly marine. We document the first Eocene bony fish and crocodyliform fossils from Banks Island.

Principal Findings

We describe fossils of bony fishes, including lepisosteid (Atractosteus), esocid (pike), and amiid, and a crocodyliform, from lower – middle Eocene strata of the Cyclic Member, Eureka Sound Formation within Aulavik National Park (∼76°N. paleolat.). Palynology suggests the sediments are late early to middle Eocene in age, and likely spanned the Early Eocene Climatic Optimum (EECO).

Conclusions/Significance

These fossils extend the geographic range of Eocene Arctic lepisosteids, esocids, amiids, and crocodyliforms west by approximately 40° of longitude or ∼1100 km. The low diversity bony fish fauna, at least at the family level, is essentially identical on Ellesmere and Banks Islands, suggesting a pan-High Arctic bony fish fauna of relatively basal groups around the margin of the Eocene Arctic Ocean. From a paleoclimatic perspective, presence of a crocodyliform, gar and amiid fishes on northern Banks provides further evidence that mild, year-round temperatures extended across the Canadian Arctic during early – middle Eocene time. Additionally, the Banks Island crocodyliform is consistent with the phylogenetic hypothesis of a Paleogene divergence time between the two extant alligatorid lineages Alligator mississippiensis and A. sinensis, and high-latitude dispersal across Beringia.     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

Auranofin disrupts selenium metabolism in Clostridium difficile by forming a stable Au–Se adduct

Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme-dependent Stickland reactions in C. difficile metabolism. In this work we have identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold-containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed on the basis of mass-spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (⁷⁵Se) in selenoproteins in both Escherichia coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin–selenide adduct in the culture medium. Addition of selenium in the form of selenite or l-selenocysteine to the growth medium significantly reduces the inhibitory action of auranofin on the growth of C. difficile. On the basis of these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Arctic plant diversity in the Early Eocene greenhouse

For the majority of the Early Caenozoic, a remarkable expanse of humid, mesothermal to temperate forests spread across Northern Polar regions that now contain specialized plant and animal communities adapted to life in extreme environments. Little is known on the taxonomic diversity of Arctic floras during greenhouse periods of the Caenozoic. We show for the first time that plant richness in the globally warm Early Eocene (approx. 55-52 Myr) in the Canadian High Arctic (76° N) is comparable with that approximately 3500 km further south at mid-latitudes in the US western interior (44-47° N). Arctic Eocene pollen floras are most comparable in richness with today's forests in the southeastern United States, some 5000 km further south of the Arctic. Nearly half of the Eocene, Arctic plant taxa are endemic and the richness of pollen floras implies significant patchiness to the vegetation type and clear regional richness of angiosperms. The reduced latitudinal diversity gradient in Early Eocene North American plant species demonstrates that extreme photoperiod in the Arctic did not limit taxonomic diversity of plants.