The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

Abstract Background Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion. Results Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the β-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the β-site was eliminated but that the active site remained otherwise intact. Conclusions Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes

Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.National Institutes of Health (HL71138, HL74167)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)INCT Obesity and DiabetesConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.Catherine E. Bond, Cheng Liu, Futoshi Kawamata, Diane M. McKeone, Winnie Fernando, Saara Jamieson, Sally-Ann Pearson, Alexandra Kane, Susan L. Woods, Tamsin R.M. Lannagan, Roshini Somashekar, Young Lee, Troy Dumenil, Gunter Hartel, Kevin J. Spring, Jennifer Borowsky, Lochlan Fennell, Mark Bettington, Jason Lee, Daniel L. Worthley, Barbara A. Leggett and Vicki L.J. Whitehal

Descriptive epidemiology of somatising tendency: findings from the CUPID study.

Somatising tendency, defined as a predisposition to worry about common somatic symptoms, is importantly associated with various aspects of health and health-related behaviour, including musculoskeletal pain and associated disability. To explore its epidemiological characteristics, and how it can be specified most efficiently, we analysed data from an international longitudinal study. A baseline questionnaire, which included questions from the Brief Symptom Inventory about seven common symptoms, was completed by 12,072 participants aged 20-59 from 46 occupational groups in 18 countries (response rate 70%). The seven symptoms were all mutually associated (odds ratios for pairwise associations 3.4 to 9.3), and each contributed to a measure of somatising tendency that exhibited an exposure-response relationship both with multi-site pain (prevalence rate ratios up to six), and also with sickness absence for non-musculoskeletal reasons. In most participants, the level of somatising tendency was little changed when reassessed after a mean interval of 14 months (75% having a change of 0 or 1 in their symptom count), although the specific symptoms reported at follow-up often differed from those at baseline. Somatising tendency was more common in women than men, especially at older ages, and varied markedly across the 46 occupational groups studied, with higher rates in South and Central America. It was weakly associated with smoking, but not with level of education. Our study supports the use of questions from the Brief Symptom Inventory as a method for measuring somatising tendency, and suggests that in adults of working age, it is a fairly stable trait

Professor of Internal Medicine

Research manuscriptNEI EY008976Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/172497/1/05 09 22 Final Tepro Supp Fig 1.pptxSEL

Thyrotropin regulates IL-6 expression in CD34+ fibrocytes: clear delineation of its cAMP-independent actions.

IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD). In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO). Recently, CD34(+) fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+) orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR), the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(-) orbital fibroblasts but reverts when CD34(+) fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt) and NF-κB (-78 to -62 nt) binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO

G protein, adenylate cyclase, and cAMP generation in orbital fibroblasts and fibrocytes.

<p>(A) Confluent cultures were treated with bTSH (5 mIU/mL), PGE₂ (1 µM), forskolin (20 µM) or nothing (control) for 16 h. Cell layers were analyzed for cAMP content and protein determination. (B) Adenylate cyclase mRNA levels were determined in orbital fibroblasts and fibrocytes from healthy donors (n = 5) and those with GD (n = 5). (C) RNA from orbital fibroblasts, fibrocytes, and thyroid tissue was subjected to RT-PCR for Gq and Gs using the C_T method. Data are expressed as mean±SD of triplicate determinations from three different strains of each. (D) Cultures indicated were treated with nothing, bTSH, PKA inhibitors H89 (10 µM), KT5720 (10 µM) or Rp-cAMP (1 mM) alone or in the combinations indicated for 6 h. RNA was extracted and subjected to RT-PCR for IL-6. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. In 3 separate experiments, H89 failed to inhibit TSH-dependent IL-6 expression (1.62±0.23-fold and 1.06±0.3-fold increase in fibroblasts and fibrocytes, respectively vs TSH alone).</p

CREB and NF-κB sites are critical to activation of the IL-6 gene promoter by bTSH.

<p>(A). Schematic demonstrating <i>cis</i>-acting regulatory elements for CREB (TGACGA, −213 to −208 nt) and NF-κB (GGGATTTTCCCA, −73 to −62 nt) relative to the transcription start site (+1). These are underlined and base substitutions resulting from site-directed mutagenesis appear above those in the corresponding wild-type sequences (emboldened). (B) Orbital fibroblasts and (C) fibrocytes were transiently transfected with empty luciferase vector or one containing the 1171 nt fragment spanning −1168 to +3 nt of the human IL-6 gene promoter, or that fragment harboring a 3 base mutation in the CREB binding site (designated “m1”), or a 4 base mutation in the NF-κB binding site (designated “m2”). Sub-confluent cultures were then treated with nothing (control) or bTSH (5 mIU/mL) for 1 h., cell layers harvested and luciferase reporter activity assessed in a luminometer. Data are expressed as the mean ± SD of triplicate determinations from a single experiment, representative of three performed. ***, P<0.001 versus TSH-treated cells transfected with wt promoter fragment. In 3 separate experiments, the m1 mutation resulted in an 86±4% and 79±4% reduction in TSH-dependent promoter activity in fibroblasts and fibrocytes respectively compared to wild type. The m2 mutation resulted in an 87±4% and 84±4% reduction, respectively.</p

bTSH induction of IL-6 is concentration- and time-dependent.

<p>(A) Orbital fibroblasts and fibrocytes were treated with escalating concentrations of bTSH for 16 h. (B) Cells were treated with bTSH (5 mIU/mL) for graded intervals indicated along the abscissas. Media were collected and subjected to ELISA or (C) IL-6 mRNA levels determined by real-time RT-PCR. Data are expressed as mean ± SD of three independent determinations. (**, p<0.01; ***, p<0.001 vs baseline). Studies were performed three times.</p

Involvement of PDK1 in the induction by TSH of IL-6.

<p>(A) Cultures were untreated (control) or bTSH (5 mIU/ml) was added to medium for 30 min. Cellular protein was subjected to Western blot analysis for PDK1 and pPDK1 and re-probed for β-actin. Results are representative of three separate experiments performed. Densitometric analysis; pPDK1, control fibroblasts, 16.25±2.85 AU; plus bTSH, 42.7±6.54 AU; control fibrocytes, 64.5±2.67 AU; plus TSH, 78.5±3.8 AU. In 3 separate experiments, TSH increased pPDK1 levels by 2.6±0.3-fold and 1.2±0.3-fold in fibroblasts and fibrocytes, respectively. (B) Sub- confluent cultures were transfected with either control siRNA or one targeting PDK1, followed by 48 h incubation. Cultures were treated with nothing or bTSH (5 mIU/mL) for 16 h, media collected and subjected to IL-6-specific ELISA and cell layers analyzed for protein content. Data are expressed as the mean ± SD of three independent determinations. Inset: Cell layers were subjected to Western blot analysis for PDK1 after transfection with control siRNA or that targeting PDK1. In 3 separate experiments, PDK1 siRNA reduced TSH-induced IL-6 levels by 73±4% in fibroblasts and 73±5% in fibrocytes. (C) Orbital fibroblasts, in this case from a patient with TAO, were transfected with PDK1siRNA while fibrocytes were treated with OSU-03012 (5 µM) for 6 h. Cultures were treated as indicated (bTSH, 5 mIU/mL) for 30 min. Cellular protein was subjected to Western blot analysis of PKCµ and pPKCµ in fibroblasts (left panel) and PKCβII and pPKCβII in fibrocytes (right panel). (D) Confluent cultures were pre-treated without or with OSU-03012 (5 µM) for 6 h, then treated with nothing (control) or bTSH (5 mIU/ml) for 30 min. Cellular proteins were subjected to Western blot analysis probing with AKT and pAKT antibodies. Inhibition of TSH-dependent pAKT by OSU-03012 in 3 separate experiments was 14.4±1.2% and 2.5±0.6% in fibroblasts and fibrocytes, respectively.</p