Capture and Characterization of Dioxygen Reactive Intermediates in CYP51 Catalysis

The cytochromes P450 (CYPs) are a superfamily of biological catalysts that are ubiquitous throughout the biological domain. CYPs are heme-b containing monooxygenases which oxidize substrates with the help of accessory redox partners. CYP substrates include endogenous compounds required for many biological functions and homeostasis, such as steroids, as well as the majority of clinically used drugs and environmental xenobiotics. The majority of studies that have been performed to date are on P450cam (CYP101) from Pseudomonas putida. Of the numerous reactions catalyzed by CYPs, unactivated carbon-carbon bond cleavage, is one of particular versatility. Being unique in their catalytic mechanisms, the C-C bond cleaving enzymes and in particular CYP51 from Mycobacterium tuberculosis are though to be capable of utilizing multiple reactive oxygen intermediates. During the process of C-C bond cleavage, CYP51 catalyzes two classical hydroxylation reactions. The final reaction culminates in an enigmatic third step which cleaves a C-C bond, liberates formate, and installs a 14,15 double bond within its steroid substrate. The mechanism of CYP51s final step is still unclear and the exact activated oxygen species has yet to be observed. CYP51 is also distinct from most CYPs owing to the fact that the acid functionality of the conserved active site “acid-alcohol pair” found in most CYPs, is replaced by a histidine. This study aimed to trap and characterize dioxygen reactive intermediates, and elucidate the role of the unique acid-alcohol pair in the formation and stabilization of these intermediates. This study demonstrates our success in generating, stabilizing, and spectroscopically characterizing reactive dioxygen intermediates in Mtb CYP51. As the life-time of the oxyferrous intermediate in Mtb CYP51 is extremely short at ambient temperatures, this work has shown the laboratory’s expertise in being able to generate reduced oxyferrous intermediates at cryogenic temperatures. These intermediates have only been generated in a handful of cytochromes P450 and as such this work adds critical information to the small body of work currently reported

Studies of Excitability in a Model Peptidergic System: The Roles of Cyclic AMP, Protein Phosphorylation and Serotonin During Afterdischarge in the Bag Cell Neurons of Aplysia californica

The polypeptide hormone-secreting bag cell neurons from the abdominal ganglion of Aplysia can be induced to fire repetitively when triggered by a brief electrical stimulus to the afferent pathway. This thesis investigates the mechanism of this afterdischarge by employing biochemical, pharmacological and electrophysiological approaches. The description of bag cell afterdischarge, its modulation by the transmitters serotonin and dopamine and evidence for the role of cyclic AMP in the genesis of afterdischarge is presented in Chapter 1. Bag cell afterdischarge is shown to be inhibited by the application of serotonin and lengthened by the application of dopamine or the methylxanthine phosphodiesterase inhibitors. Cyclic AMP undergoes a 2-3 fold increase in the bag cell clusters during an electrically-stimulated afterdischarge but not in matched controls where equivalent electrical stimulation did not elicit afterdischarge. As further evidence for a role for cyclic AMP in the genesis of afterdischarge, afterdischarges were obtained in unstimulated preparations by the extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP and 8-methylthio-cyclic AMP. Chapter 2 describes protein phosphorylation in bag cell tissues under a number of different conditions. The presence of an endogenous, cyclic AMP-dependent protein kinase activity is demonstrated in crude membranes prepared from bag cells and the substrate specificity for this activity is shown to be similar to that of protein kinase catalytic subunit prepared from bovine heart. Increases in phosphorylation of a 33,000 dalton and 21,000 dalton phosphoprotein are shown to occur during electrically-stimulated afterdischarge in bag cells. The 21,000 dalton substrate is shown to be apparently specific to bag cell tissues and an amino acid composition and partial amino acid sequence of this protein is presented. Chapter 3 presents evidence that serotonin, within the physiological range reported by other workers for Aplysia (0.1-1.0 μM) brings about a rapid inhibition of an ongoing afterdischarge. This inhibition is antagonized by the stereospecific blocker of serotonin action, D-butaclamol but not its inactive isomer, L-butaclamol. Serotonergic inhibition is shown to be associated with decreased bag cell action potential duration and height and an increased threshold to spike generation. Evidence is presented that the second, calcium-dependent phase of bag cell afterdischarge is most sensitive to the action of the transmitter and that the potassium channel blocker, tetraethylammonium can overcome serotonin's inhibitory effect. This raises the possibility that serotonin may cause inhibition of bag cell afterdischarge by increasing potassium conductance. The possible functional role of serotonin inhibition of egg-laying is discussed.</p

Party identification and party closeness in comparative perspective

The present analysis uses data from 1974 and 1981 U. S. cross sections, which incorporate a panel, to compare the standard NES measure of party identification (ID) with a measure of partisanship derived from a party closeness question widely employed in cross-national research. Important features of the two scales are examined by transforming the closeness measure into a scale of very close, fairly close, not very close, and no preference corresponding to the seven-point ID scale. The scales are highly correlated and are similar in their reliability. More than 75% of the “independents” in the ID scale choose a party in the closeness version, and over half of these select the “fairly close” category. Respondents do not volunteer that they are independents when that alternative is not stated in the question.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45482/1/11109_2004_Article_BF00990552.pd

American political affiliation, 2003–43: a cohort component projection

The recent rise and stability in American party identification has focused interest on the long-term dynamics of party bases. Liberal commentators cite immigration and youth as forces which will produce a natural Democratic advantage in the future while conservative writers highlight the importance of high Republican fertility in securing Republican growth. These concerns foreground the neglect of demography within political science. This paper addresses this omission by conducting the first ever cohort component projection of American partisan populations to 2043 based on survey and census data. A number of scenarios are modeled, but, on current trends, we predict that American partisanship will shift much less than the nation’s ethnic composition because the parties’ age structures are similar. Still, our projections find that the Democrats gain two to three percentage points from the Republicans by 2043, mainly through immigration, though Republican fertility may redress the balance in the very long term

The origins of redistributive policy preferences: political socialisation with and without a welfare state

Research on the impact of the macroeconomy on individual-level preferences for redistribution has produced varying results. This paper presents a new theory on the presence of an expansive welfare state during one’s formative years as a source of heterogeneity in the effect that macroeconomic conditions have on individuals’ preferences for redistributive policy. This theory is tested using cohort analysis via the British Social Attitudes surveys (1983–2010), with generations coming of age between the end of World War I and today. Findings confirm that cohorts that were socialised before and after the introduction of the welfare state react differently to economic crises: the former become less supportive of redistribution, while the latter become more supportive. The research sheds light on the long-term shifts of support for the welfare state due to generational replacement

Sexual selection on song and cuticular hydrocarbons in two distinct populations of Drosophila montana

Sexual selection has the potential to contribute to population divergence and speciation. Most studies of sexual selection in Drosophila have concentrated on a single signaling modality, usually either courtship song or cuticular hydrocarbons (CHCs), which can act as contact pheromones. We have examined the relationship between both signal types and reproductive success using F1–3 offspring of wild- collected flies, raised in the lab. We used two populations of the Holarctic species Drosophila montana that represent different phylogeographic clades that have been separate for ca. 0.5 million years (MY), and differ to some extent in both traits. Here, we characterize the nature and identify the targets of sexual selection on song, CHCs, and both traits combined within the populations. Three measures of courtship outcome were used as fitness proxies. They were the probability of mating, mating latency, and the production of rejection song by females, and showed patterns of association with different traits that included both linear and quadratic selection. Courtship song predicted courtship outcome better than CHCs and the signal modalities acted in an additive rather than synergistic manner. Selection was generally consistent in direction and strength between the two populations and favored males that sang more vigorously. Sexual selection differed in the extent, strength, and nature on some of the traits between populations. However, the differences in the directionality of selection detected were not a good predictor of population differences. In addition, a character previously shown to be important for species recognition, interpulse interval, was found to be under sexual selection. Our results highlight the complexity of understanding the relationship between within-population sexual selection and population differences. Sexual selection alone cannot predict differences between populations.Publisher PDFPeer reviewe

Procalcitonin and Other Common Biomarkers Do Not Reliably Identify Patients at Risk for Bacterial Infection After Congenital Heart Surgery

Objectives: Following surgery, it is difficult to distinguish a postoperative inflammatory reaction from infection. This study examined the predictive value of the biomarkers; procalcitonin, C-reactive protein, lactate, neutrophils, lymphocytes, platelets, and the biphasic activated partial thromboplastin time waveform in diagnosing bacterial infection following cardiac surgery. Design: Prospective, observational study. Setting: A regional, PICU in the United Kingdom. Patients: Three-hundred sixty-eight children under the age of 16 admitted to the PICU for elective cardiac surgery were enrolled in the study. Interventions: All biomarker measurements were determined daily until postoperative day 7. Children were assessed for postoperative infection until day 28 and divided into four groups: bacterial infection, culture-negative sepsis, viral infection, and no infection. We used the Kruskal-Wallis test, chi-square test, analysis of variance, and area under the curve in our analysis. Measurements and Main Results: In total, 71 of 368 children (19%) developed bacterial infection postoperatively, the majority being surgical site infections. In those with bacterial infection, procalcitonin was elevated on postoperative days 1–3 and the last measurement prior to event compared with those without bacterial infection. The most significant difference was the last measurement prior to event; 0.72 ng/mL in the bacterial infection group versus 0.13 ng/mL in the no infection group (for all groups; p and#60; 0.001). Longitudinal profiles of all biomarkers were indistinct in the bacterial infection and nonbacterial infection groups except in those with culture-negative infections who had distinct procalcitonin kinetics on postoperative days 1–4. Children with culture-negative sepsis required longer ventilatory support and PICU stay and were more likely to develop complications than the other groups. Conclusions: None of the biomarkers studied within 3 days of infection distinguished between infection and postoperative inflammatory reaction. However, procalcitonin kinetics peaked on postoperative day 2 and fell more sharply than C-reactive protein kinetics, which peaked at postoperative day 3. The monitoring of procalcitonin kinetics following cardiac surgery may help guide rational antimicrobial use

Exploring Late Bronze Age systems of bronzework production in Switzerland through Network Science

YesMany hundreds of Bronze Age bronze artefacts are known from excavations in Switzerland, yet the interpretation of production networks from the object find locations remain problematic. It is proposed that the decorative elements used on items, such as ring-jewellery, can be used as elements to assist in the identification of artisanal traditions and ‘schools’, and also regional or community preference and selection of specific designs. Combining the analysis of over 1700 items of ring-jewellery from Switzerland with approaches from network science has facilitated the identification of regional clustering of design elements, comparable with cultural typologies in the area. It is also possible to identify potential instances of cultural differentiation through decoration within the broader regional cultural traditions. The study highlights important facets of bronzework production in the region of Switzerland, while also demonstrating future potential directions which could build upon the European wide dataset of prehistoric bronzework.Primary research conducted under previous funding at University of Basel, Switzerland – SNF gran