supplement(1) – Supplemental material for The Role of Constitutional Features in Judicial Review

<p>Supplemental material, supplement(1) for The Role of Constitutional Features in Judicial Review by Adam R. Brown in State Politics & Policy Quarterly</p

Enantioselective Thiourea-Catalyzed Intramolecular Cope-Type Hydroamination

Catalysis of Cope-type rearrangements of bis-homoallylic hydroxylamines is demonstrated using chiral thiourea derivatives. This formal intramolecular hydroamination reaction provides access to highly enantioenriched α-substituted pyrrolidine products and represents a complementary approach to metal-catalyzed methods

A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

<div><p>Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant <i>Staphylococcus aureus</i>. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical <i>Hydrastis Canadensis</i>, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC₅₀ value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC₅₀ values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.</p></div

Efflux pump inhibition in <i>S</i>. <i>aureus</i> by a goldenseal (<i>Hydrastis canadensis</i>) extract.

<p>(A) Data collected using the fluorescence-based ethidium accumulation assay for a range of <i>H</i>. <i>canadensis</i> extract concentrations. (B) Data collected using the mass spectrometry-based ethidium accumulation assay. Incubation time was 30 min for both A and B, data represents mean of 3 samples, error bars represent standard deviation.</p

Efflux pump inhibitory activity of apigenin, piperine, and quercetin as indicated by LC-MS measurement of residual ethidium bromide in spent broth after a 30 min incubation.

<p>Relative peak area (expressed as a percentage) for ethidium is plotted as a function of concentration of the putative inhibitor. Data points represent the mean of 3 measurements (biological replicates), with error bars representing standard deviation.</p

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

<div><p>The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (<i>Fasn, Srebf1</i>) and cholesterol biosynthetic (<i>Hmgcsr</i>, <i>Hmgcs1</i>), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (<i>Irs1</i>, <i>Irs2, Prkce</i>) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.</p></div

Efflux pump inhibitory activity and antimicrobial activity of flavonoids.

<p>a: Efflux pump inhibition was measured via LC-MS analysis of ethidium in spent, filtered culture supernatant after a 30 min incubation in triplicate wells.</p><p>b: Growth inhibition was measured by optical density at 600nm (in triplicate) after an 18 hr incubation.</p><p>c: CCCP is an abbreviation for the compound carbonyl cyanide m-chloro-phenylhydrazone</p><p>Efflux pump inhibitory activity and antimicrobial activity of flavonoids.</p

ME1-Tg mice on chow diet exhibit increased ME1 protein and mRNA abundance in small intestine.

<p>A) Schematic representation of the mouse villin1-ME1 transgene construct in which a complete open reading frame for rat ME1 was placed downstream of the murine villin1 gene promoter-enhancer (12.4 kb fragment). The SV40 polyA signal-containing region was located downstream of the <i>Me1</i> cDNA sequence. Red arrows indicate the location of genotyping primers, while black arrows indicate the location of primers used to detect transgene expression by RT-PCR. B) ME1-Tg mRNA expression in the jejunum and colon of WT and ME1-Tg mice detected by RT-PCR (n = 3 mice/group; Exp.1). C) Representative Western blots of ME1 protein in the jejunum, ileum and colon of WT and ME1-Tg mice (n = 2−3/group; Exp. 1). The ME1 antibody detected endogenous murine and Tg-derived rat ME1 proteins. D) Densitometric analysis of relative ME1 protein levels in panel C. E) Representative images of immunohistochemical staining of ME1 in the Ileum and colon of WT and ME1-Tg Tg mice. Scale bars = 100 µM. Arrows indicate villous epithelial and luminal epithelial staining of ME1 in the ileum and colon, respectively. F) Weight gain calculated as percentage increase of final body weight from initial body weight of WT and ME1-Tg male mice (n = 8−10 mice/group) from Exp. 1. Bar graphs represent mean ± SEM; *Significant difference at <i>P</i><0.05 between genotypes. **Significant difference at <i>P</i><0.01 between genotypes. <i>P</i> values are indicated for those tending to have significant differences between groups (0.05<<i>P</i><0.10).</p

Change in absolute fluorescent intensity over time for <i>Staphylococcus aureus</i> exposed to ethidium bromide in the efflux pump inhibitor piperine.

<p>Fluorescence increases over time due to intracellular accumulation of ethidium bromide. The increase is more pronounced in the presence of piperine, which enhances intracellular accumulation of ethidium bromide by blocking efflux. Data points represent the mean of 3 samples, error bars represent standard deviation.</p