Publications by authors named "Jan R Crowley"

37 Publications

Astrocyte deletion of α2-Na/K ATPase triggers episodic motor paralysis in mice via a metabolic pathway.

Nat Commun 2020 12 2;11(1):6164. Epub 2020 Dec 2.

Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.
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http://dx.doi.org/10.1038/s41467-020-19915-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710756PMC
December 2020

Author Correction: High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

Nat Metab 2020 09;2(9):991

Department of Medicine, Cardiovascular Division, Washington University School of Medicine, St Louis, MO, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s42255-020-00291-6DOI Listing
September 2020

High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

Nat Metab 2020 01 23;2(1):110-125. Epub 2020 Jan 23.

Department of Medicine, Cardiovascular Division, Washington University School of Medicine, St Louis, MO, USA.

High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.
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http://dx.doi.org/10.1038/s42255-019-0162-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053091PMC
January 2020

Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice.

Gastroenterology 2020 04 12;158(5):1402-1416.e2. Epub 2019 Dec 12.

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Background & Aims: Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile.

Methods: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses.

Results: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection.

Conclusions: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1053/j.gastro.2019.11.295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103499PMC
April 2020

Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections.

J Clin Invest 2019 08 12;129(9):3792-3806. Epub 2019 Aug 12.

Center for Women's Infectious Disease Research, Division of Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non-C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.
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http://dx.doi.org/10.1172/JCI126905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715368PMC
August 2019

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis.

Nat Commun 2017 06 7;8:15750. Epub 2017 Jun 7.

Department of Medicine, Cardiovascular Division, Washington University School of Medicine, Campus Box 8086, 660 S Euclid Avenue, St Louis, Missouri 63110, USA.

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease.
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http://dx.doi.org/10.1038/ncomms15750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467270PMC
June 2017

SLC2A8 (GLUT8) is a mammalian trehalose transporter required for trehalose-induced autophagy.

Sci Rep 2016 12 6;6:38586. Epub 2016 Dec 6.

Department of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.

Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.
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http://dx.doi.org/10.1038/srep38586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138640PMC
December 2016

Human Metabolome-derived Cofactors Are Required for the Antibacterial Activity of Siderocalin in Urine.

J Biol Chem 2016 Dec 25;291(50):25901-25910. Epub 2016 Oct 25.

From the Division of Infectious Diseases, Department of Medicine,

In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function.
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http://dx.doi.org/10.1074/jbc.M116.759183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207064PMC
December 2016

Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

PLoS One 2016 20;11(7):e0159675. Epub 2016 Jul 20.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159675PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954648PMC
July 2017

Urinary Metabolomics Identifies a Molecular Correlate of Interstitial Cystitis/Bladder Pain Syndrome in a Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network Cohort.

EBioMedicine 2016 May 31;7:167-74. Epub 2016 Mar 31.

Center for Women's Infectious Diseases Research, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Division of Infectious Diseases, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Department of Internal Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, United States. Electronic address:

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a poorly understood syndrome affecting up to 6.5% of adult women in the U.S. The lack of broadly accepted objective laboratory markers for this condition hampers efforts to diagnose and treat this condition. To identify biochemical markers for IC/BPS, we applied mass spectrometry-based global metabolite profiling to urine specimens from a cohort of female IC/BPS subjects from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network. These analyses identified multiple metabolites capable of discriminating IC/BPS and control subjects. Of these candidate markers, etiocholan-3α-ol-17-one sulfate (Etio-S), a sulfoconjugated 5-β reduced isomer of testosterone, distinguished female IC/BPS and control subjects with a sensitivity and specificity >90%. Among IC/BPS subjects, urinary Etio-S levels are correlated with elevated symptom scores (symptoms, pelvic pain, and number of painful body sites) and could resolve high- from low-symptom IC/BPS subgroups. Etio-S-associated biochemical changes persisted through 3-6months of longitudinal follow up. These results raise the possibility that an underlying biochemical abnormality contributes to symptoms in patients with severe IC/BPS.
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http://dx.doi.org/10.1016/j.ebiom.2016.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909380PMC
May 2016

Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis.

Sci Signal 2016 Feb 23;9(416):ra21. Epub 2016 Feb 23.

Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.
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http://dx.doi.org/10.1126/scisignal.aac5472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816640PMC
February 2016

Deconvoluting heme biosynthesis to target blood-stage malaria parasites.

Elife 2015 Jul 14;4. Epub 2015 Jul 14.

Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States.

Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.
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http://dx.doi.org/10.7554/eLife.09143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532139PMC
July 2015

Human Urinary Composition Controls Antibacterial Activity of Siderocalin.

J Biol Chem 2015 Jun 10;290(26):15949-60. Epub 2015 Apr 10.

From the Division of Infectious Diseases, Department of Medicine, Center for Women's Infectious Disease Research, and

During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.
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http://dx.doi.org/10.1074/jbc.M115.645812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481200PMC
June 2015

Metal selectivity by the virulence-associated yersiniabactin metallophore system.

Metallomics 2015 Jun;7(6):1011-22

Center for Women's Infectious Diseases Research, Washington University School of Medicine, Box 8051, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA.

Uropathogenic Escherichia coli secrete siderophores during human infections. Although siderophores are classically defined by their ability to bind iron(III) ions, the virulence-associated siderophore yersiniabactin was recently found to bind divalent copper ions during urinary tract infections. Here we use a mass spectrometric approach to determine the extent of non-iron(III) metal interactions by yersiniabactin and its TonB-dependent outer membrane importer FyuA. In addition to copper, iron and gallium ions, yersiniabactin was also observed to form stable nickel, cobalt, and chromium ion complexes. In E. coli, copper(II) and all other non-iron(III) yersiniabactin complexes were imported by FyuA in a TonB-dependent manner. Among metal-yersiniabactin complexes, copper(II) yersiniabactin is predicted to be structurally distinctive and was the only complex not to competitively inhibit iron(III) yersiniabactin import. These results are consistent with yersiniabactin as part of a metallophore system able to prioritize iron(III) complex uptake in high copper environments.
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http://dx.doi.org/10.1039/c4mt00341aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809022PMC
June 2015

Malaria parasites produce volatile mosquito attractants.

mBio 2015 Mar 24;6(2). Epub 2015 Mar 24.

Unlabelled: The malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid organelle that possesses plant-like metabolic pathways. Plants use the plastidial isoprenoid biosynthesis pathway to produce volatile odorants, known as terpenes. In this work, we describe the volatile chemical profile of cultured malaria parasites. Among the identified compounds are several plant-like terpenes and terpene derivatives, including known mosquito attractants. We establish the molecular identity of the odorant receptors of the malaria mosquito vector Anopheles gambiae, which responds to these compounds. The malaria parasite produces volatile signals that are recognized by mosquitoes and may thereby mediate host attraction and facilitate transmission.

Importance: Malaria is a key global health concern. Mosquitoes that transmit malaria are more attracted to malaria parasite-infected mammalian hosts. These studies aimed to understand the chemical signals produced by malaria parasites; such an understanding may lead to new transmission-blocking strategies or noninvasive malaria diagnostics.
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http://dx.doi.org/10.1128/mBio.00235-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453533PMC
March 2015

The heme biosynthesis pathway is essential for Plasmodium falciparum development in mosquito stage but not in blood stages.

J Biol Chem 2014 Dec 28;289(50):34827-37. Epub 2014 Oct 28.

From the Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129,

Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
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http://dx.doi.org/10.1074/jbc.M114.615831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263882PMC
December 2014

Escherichia coli biofilms have an organized and complex extracellular matrix structure.

mBio 2013 Sep 10;4(5):e00645-13. Epub 2013 Sep 10.

Center for Women's Infectious Diseases Research, Washington University School of Medicine, St. Louis, Missouri, USA.

Unlabelled: Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community.

Importance: Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.
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http://dx.doi.org/10.1128/mBio.00645-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774191PMC
September 2013

Direct tests of enzymatic heme degradation by the malaria parasite Plasmodium falciparum.

J Biol Chem 2012 Nov 19;287(45):37793-807. Epub 2012 Sep 19.

Department of Molecular Microbiology and the Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.
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http://dx.doi.org/10.1074/jbc.M112.414078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488054PMC
November 2012

The siderophore yersiniabactin binds copper to protect pathogens during infection.

Nat Chem Biol 2012 Aug 8;8(8):731-6. Epub 2012 Jul 8.

Center for Women's Infectious Diseases Research, Washington University School of Medicine, St. Louis, MO, USA.

Bacterial pathogens secrete chemically diverse iron chelators called siderophores, which may exert additional distinctive functions in vivo. Among these, uropathogenic Escherichia coli often coexpress the virulence-associated siderophore yersiniabactin (Ybt) with catecholate siderophores. Here we used a new MS screening approach to reveal that Ybt is also a physiologically favorable Cu(II) ligand. Direct MS detection of the resulting Cu(II)-Ybt complex in mice and humans with E. coli urinary tract infections demonstrates copper binding to be a physiologically relevant in vivo interaction during infection. Ybt expression corresponded to higher copper resistance among human urinary tract isolates, suggesting a protective role for this interaction. Chemical and genetic characterization showed that Ybt helps bacteria resist copper toxicity by sequestering host-derived Cu(II) and preventing its catechol-mediated reduction to Cu(I). Together, these studies reveal a new virulence-associated function for Ybt that is distinct from iron binding.
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http://dx.doi.org/10.1038/nchembio.1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600419PMC
August 2012

Postprandial leucine and insulin responses and toxicological effects of a novel whey protein hydrolysate-based supplement in rats.

J Int Soc Sports Nutr 2012 Jun 6;9(1):24. Epub 2012 Jun 6.

Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

The purpose of this study was: aim 1) compare insulin and leucine serum responses after feeding a novel hydrolyzed whey protein (WPH)-based supplement versus a whey protein isolate (WPI) in rats during the post-absorptive state, and aim 2) to perform a thorough toxicological analysis on rats that consume different doses of the novel WPH-based supplement over a 30-day period. In male Wistar rats (~250 g, n = 40), serum insulin and leucine concentrations were quantified up to 120 min after one human equivalent dose of a WPI or the WPH-based supplement. In a second cohort of rats (~250 g, n = 20), we examined serum/blood and liver/kidney histopathological markers after 30 days of feeding low (1human equivalent dose), medium (3 doses) and high (6 doses) amounts of the WPH-based supplement. In aim 1, higher leucine levels existed at 15 min after WPH vs. WPI ingestion (p = 0.04) followed by higher insulin concentrations at 60 min (p = 0.002). In aim 2, liver and kidney histopathology/toxicology markers were not different 30 days after feeding with low, medium, high dose WPH-based supplementation or water only. There were no between-condition differences in body fat or lean mass or circulating clinical chemistry markers following the 30-day feeding intervention in aim 2. In comparison to WPI, acute ingestion of a novel WPH-based supplement resulted in a higher transient leucine response with a sequential increase in insulin. Furthermore, chronic ingestion of the tested whey protein hydrolysate supplement appears safe.
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http://dx.doi.org/10.1186/1550-2783-9-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404932PMC
June 2012

Substrate metabolism during basal and hyperinsulinemic conditions in adolescents and young-adults with Barth syndrome.

J Inherit Metab Dis 2013 Jan 12;36(1):91-101. Epub 2012 May 12.

Program in Physical Therapy & Department of Medicine, Washington University School of Medicine, Box 8502, St Louis, MO 63108, USA.

Background: Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by mitochondrial abnormalities, infantile or childhood onset of cardioskeletal myopathy, and high mortality rates. It is currently unknown if BTHS related mitochondrial dysfunction results in substrate metabolism abnormalities and thereby contributes to cardioskeletal myopathy in patients with BTHS.

Methods: Adolescents and young adults with BTHS (n = 5, 20 ± 4 yrs) and age and activity matched healthy controls (n = 5, 18 ± 4 yrs) underwent an hyperinsulinemic-euglycemic clamp procedure with stable isotopically labeled tracers for measurement of lipolysis, fatty acid oxidation, glucose disposal, and whole-body proteolysis rates; dual energy x-ray absorptiometry for measurement of body composition and 2-D and strain echocardiography for measurement of left ventricular function.

Results: Participants with BTHS had lower fat-free mass (FFM) (BTHS: 31.4 ± 6.9 vs.

Control: 46.7 ± 5.3 kg, p < 0.005), lower systolic function (strain, BTHS: -15.2 ± 2.4 vs.

Control: -19.0 ± 2.4 %, p < 0.05), greater insulin-stimulated glucose disposal rate per kg FFM (BTHS: 96.5 ± 16.3 vs.

Control: 67.4 ± 17.6 μmol/kgFFM/min, p < 0.05), lower basal (BTHS: 4.6 ± 2.7 vs.

Control: 11.9 ± 4.4 μmol/kgFM/min, p < 0.05) and hyperinsulinemic (BTHS: 1.6 ± 0.4 vs.

Control: 3.6 ± 1.6 μmol/kgFM/min, p < 0.05) lipolytic rate per kg fat mass (FM), and a trend towards higher basal leucine rate of appearance per kg FFM (BTHS: 271.4 ± 69.3 vs.

Control: 193.1 ± 28.7 μmol/kgFFM/hr, p = 0.07) compared to controls. Higher basal leucine rate of appearance per kg FFM (i.e. whole-body proteolytic rate) tended to be associated with lower left ventricular systolic strain (r = -0.57, p = 0.09).

Conclusion: Whole-body fatty acid, glucose and amino acid metabolism kinetics when expressed per unit of body composition are altered and appear to be related to cardioskeletal myopathy in humans with BTHS. Further studies examining myocardial substrate metabolism and whole-body substrate metabolism during increased energy demands (e.g., exercise) and their relationships to skeletal and cardiac function are recommended.
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http://dx.doi.org/10.1007/s10545-012-9486-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608431PMC
January 2013

Treatment and prevention of urinary tract infection with orally active FimH inhibitors.

Sci Transl Med 2011 Nov;3(109):109ra115

Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

Chronic and recurrent urinary tract infections pose a serious medical problem because there are few effective treatment options. Patients with chronic urinary tract infections are commonly treated with long-term prophylactic antibiotics that promote the development of antibiotic-resistant forms of uropathogenic Escherichia coli (UPEC), further complicating treatment. We developed small-molecular weight compounds termed mannosides that specifically inhibit the FimH type 1 pilus lectin of UPEC, which mediates bacterial colonization, invasion, and formation of recalcitrant intracellular bacterial communities in the bladder epithelium. Here, we optimized these compounds for oral bioavailability and demonstrated their fast-acting efficacy in treating chronic urinary tract infections in a preclinical murine model. These compounds also prevented infection in vivo when given prophylactically and strongly potentiated the activity of the current standard of care therapy, trimethoprim-sulfamethoxazole, against clinically resistant PBC-1 UPEC bacteria. These compounds have therapeutic efficacy after oral administration for the treatment of established urinary tract infections in vivo. Their unique mechanism of action-targeting the pilus tip adhesin FimH-circumvents the conventional requirement for drug penetration of the outer membrane, minimizing the potential for the development of resistance. The small-molecular weight compounds described herein promise to provide substantial benefit to women suffering from chronic and recurrent urinary tract infections.
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http://dx.doi.org/10.1126/scitranslmed.3003021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694776PMC
November 2011

Regulation of myocardial ketone body metabolism by the gut microbiota during nutrient deprivation.

Proc Natl Acad Sci U S A 2009 Jul 22;106(27):11276-81. Epub 2009 Jun 22.

Center for Genome Sciences and Department of Medicine, Washington University School of Medicine, St. Louis, MO 63108, USA.

Studies in mice indicate that the gut microbiota promotes energy harvest and storage from components of the diet when these components are plentiful. Here we examine how the microbiota shapes host metabolic and physiologic adaptations to periods of nutrient deprivation. Germ-free (GF) mice and mice who had received a gut microbiota transplant from conventionally raised donors were compared in the fed and fasted states by using functional genomic, biochemical, and physiologic assays. A 24-h fast produces a marked change in gut microbial ecology. Short-chain fatty acids generated from microbial fermentation of available glycans are maintained at higher levels compared with GF controls. During fasting, a microbiota-dependent, Ppar alpha-regulated increase in hepatic ketogenesis occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of heart rate, hydraulic work, and output, mitochondrial morphology, number, and respiration, plus ketone body, fatty acid, and glucose oxidation in isolated perfused working hearts from GF and colonized animals (combined with in vivo assessments of myocardial physiology) revealed that the fasted GF heart is able to sustain its performance by increasing glucose utilization, but heart weight, measured echocardiographically or as wet mass and normalized to tibial length or lean body weight, is significantly reduced in both fasted and fed mice. This myocardial-mass phenotype is completely reversed in GF mice by consumption of a ketogenic diet. Together, these results illustrate benefits provided by the gut microbiota during periods of nutrient deprivation, and emphasize the importance of further exploring the relationship between gut microbes and cardiovascular health.
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http://dx.doi.org/10.1073/pnas.0902366106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700149PMC
July 2009

The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts.

Arch Ophthalmol 2009 Apr;127(4):475-82

Department of Ophthalmology and Visual Sciences, Washington University, St Louis, Missouri 63110, USA.

Objective: To investigate the rate and mechanism of oxygen consumption by the vitreous.

Methods: Oxygen consumption was measured with a microrespirometer. Vitreous ascorbate was measured spectrophotometrically and by gas chromatography-mass spectrometry. Vitreous degeneration was related to the rate of oxygen consumption and ascorbate concentration in samples obtained during vitrectomy.

Results: Prolonged exposure to oxygen or treatment with ascorbate oxidase eliminated oxygen consumption by the vitreous. Adding ascorbate restored oxygen consumption. Oxygen consumption persisted after boiling or treating the vitreous with the chelating agents EDTA and deferoxamine. In patients undergoing retinal surgery, liquefaction of the vitreous and previous vitrectomy were associated with decreased ascorbate concentration and lower oxygen consumption.

Conclusions: Ascorbate in the vitreous decreases exposure of the lens to oxygen. The catalyst for this reaction is not known, although free iron may contribute. The gel state of the vitreous preserves ascorbate levels, thereby sustaining oxygen consumption. Vitrectomy or advanced vitreous degeneration may increase exposure of the lens to oxygen, promoting the progression of nuclear cataracts.

Clinical Relevance: Determining how the eye is protected from nuclear cataracts should suggest treatments to reduce their incidence.
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http://dx.doi.org/10.1001/archophthalmol.2008.621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683478PMC
April 2009

Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli.

PLoS Pathog 2009 Feb 20;5(2):e1000305. Epub 2009 Feb 20.

Center for Women's Infectious Diseases Research, Washington University School of Medicine, St Louis, MO, USA.

Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.
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http://dx.doi.org/10.1371/journal.ppat.1000305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2637984PMC
February 2009

Tetrahydrofurandiols (THF-diols), leukotoxindiols (LTX-diols), and endocrine disruption in rats.

Environ Health Perspect 2007 May 29;115(5):702-8. Epub 2007 Jan 29.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Background: Ground corncob animal bedding and corn food products contain substances that disrupt endocrine function in rats. The disruptors were identified as isomeric mixtures of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid) and leukotoxindiols (LTX-diols; 9,10-dihydroxy-12-octadecenoic acid and 12,13-dihydroxy-9-octadecenoic acid). The authentic compounds blocked sexual behavior in male rats and estrous cyclicity in female rats at oral doses of 2 ppm.

Objectives: To define the lowest observed adverse effect level (LOAEL) for the THF-diols and LTX-diols in rats, we examined the nature of their interaction (additive or synergistic) and quantified the concentration of THF-diols in rat tissues.

Methods: Adult male and female rats were provided drinking solutions containing various doses of THF-diols and/or LTX-diols, and we evaluated their effects on male sexual behavior and female estrous cyclicity. Tissues were collected for THF-diol determination by gas chromatography-mass spectrometry.

Results: The LOAEL for THF-diols and LTX-diols for blocking estrous cyclicity was 0.5-1.0 ppm and 0.2-0.5 ppm, respectively. Higher concentrations (1-2 ppm) of THF-diols were required to block male sexual behavior. Combination studies with subthreshold doses of 0.05 ppm THF-diols plus 0.05 ppm LTX-diols revealed that their effects on estrous cyclicity were not synergistic. We were unable to detect THF-diols in tissues from rats treated with 10 ppm of the compounds, suggesting that metabolism may be involved.

Discussion: THF-diols, LTX-diols, and/or their metabolites likely act additively to disrupt endocrine function in male and female rats at concentrations (0.5-1 ppm) that are 200-fold lower than those of classical phytoestrogen endocrine disruptors.
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http://dx.doi.org/10.1289/ehp.9311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868002PMC
May 2007

Leukotoxin diols from ground corncob bedding disrupt estrous cyclicity in rats and stimulate MCF-7 breast cancer cell proliferation.

Environ Health Perspect 2005 Dec;113(12):1698-704

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (iso-leukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1314908PMC
http://dx.doi.org/10.1289/ehp.8231DOI Listing
December 2005

An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers.

Proc Natl Acad Sci U S A 2005 Jul 5;102(28):9901-6. Epub 2005 Jul 5.

Center for Genome Sciences and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Human neuroendocrine (NE) cancers range from relatively indolent to highly aggressive. In this study, we combine functional genomics with metabolomics to identify features of NE cancers associated with a poor outcome. Analysis of GeneChip datasets of primary prostate tumors, as well as lymph node and liver metastases from transgenic mice with a NE cell cancer, plus derived NE cell lines yielded a signature of 446 genes whose expression is enriched in neoplastic mouse prostatic NE cells. This signature was used for in silico metabolic reconstructions of NE cell metabolism, directed liquid chromatography/tandem MS analysis of metabolites in prostatic NE tumors and cell lines, and analysis of GeneChip datasets of human NE tumors with good or poor prognoses. The results indicate that a distinguishing feature of poor-prognosis NE tumors is a glutamic acid decarboxylase-independent pathway for production of GABA and a pathway for production of imidazole-4-acetate that involves dopa decarboxylase and a membrane-associated amine oxidase, amiloride-binding protein 1. Electrophysiological studies disclosed that imidazole-4-acetate can bind and activate GABA(A) receptors expressed by transformed NE cells, thus providing a previously uncharacterized paradigm for NE tumor cell signaling. Transcriptional, metabolic, and electrophysiologic features of transformed mouse NE cells are also evident in neural progenitor cells.
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http://dx.doi.org/10.1073/pnas.0500756102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1169236PMC
July 2005

Nuclear type II [3H]estradiol binding sites: a histone H3-H4 complex.

J Steroid Biochem Mol Biol 2005 Jun;96(1):19-30

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA.

[(3)H]luteolin covalently labels two forms (11kDa and 35kDa proteins) of type II binding sites in rat uterine nuclear extracts [K. Shoulars, T. Brown, M. Alejandro, J. Crowley, B. Markaverich, Identification of rat uterine nuclear type II [(3)H]estradiol binding sites as histone H4, Biochem. Biophys. Res. Commun. 296 (2002) 1083-1090]. The 11kDa protein was identified as histone H4. Levels of the 35kDa protein were insufficient for sequencing; however, this protein was recognized by anti-histone H4 antibodies. Histones H3 and H4 exist as dimers in vivo (mw>35kDa) and we suspected the 35kDa [(3)H]luteolin-labeled protein in uterine nuclear extracts might be a complex of histones H3 and H4. This manuscript describes methods for the purification of commercially available calf thymus core histones that retain [(3)H]luteolin binding activity and are of sufficient purity for recombination studies. Mixing experiments with pure H3 and H4 from calf thymus demonstrate that a 35kDa H3-H4 dimer capable of binding [(3)H]luteolin is generated and this protein appears equivalent to the 35kDa [(3)H]luteolin binding protein in rat uterine nuclear extracts. If this is the case, type II site ligands including MeHPLA, luteolin, and other bioflavonoids and phytoestrogens may control histone-dependent gene transcription and cellular proliferation via binding to and modulating core histone/nucleosome function.
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http://dx.doi.org/10.1016/j.jsbmb.2004.12.047DOI Listing
June 2005

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease.

J Neurochem 2004 Aug;90(3):724-33

Department of Medicine, University of Washington, Seattle, Washington 98195, USA.

Myeloperoxidase, a heme protein expressed by professional phagocytic cells, generates an array of oxidants which are proposed to contribute to tissue damage during inflammation. We now report that enzymatically active myeloperoxidase and its characteristic amino acid oxidation products are present in human brain. Further, expression of myeloperoxidase is increased in brain tissue showing Alzheimer's neuropathology. Consistent with expression in phagocytic cells, myeloperoxidase immunoreactivity was present in some activated microglia in Alzheimer brains. However, the majority of immunoreactive material in brain localized with amyloid plaques and, surprisingly, neurons including granule and pyramidal neurons of the hippocampus. Confirming neuronal localization of the enzyme, several neuronal cell lines as well as primary neuronal cultures expressed myeloperoxidase protein. Myeloperoxidase mRNA was also detected in neuronal cell lines. These results reveal the unexpected presence of myeloperoxidase in neurons. The increase in neuronal myeloperoxidase expression we observed in Alzheimer disease brains raises the possibility that the enzyme contributes to the oxidative stress implicated in the pathogenesis of the neurodegenerative disorder.
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http://dx.doi.org/10.1111/j.1471-4159.2004.02527.xDOI Listing
August 2004