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Ann Arbor, MI, United States

Artymovich K.,Essen BioScience | Kim J.-S.,Korea Food Research Institute | Linz J.E.,Michigan State University | Hall D.F.,Neogen Corporation | And 5 more authors.
Food Microbiology | Year: 2013

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific " successful alleles" at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a " successful" Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the " unsuccessful" Cj0045 11G allele experienced phase variation to " successful" 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the " successful" 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice. © 2013 Elsevier Ltd.

Pier D.M.,University of Leicester | Pier D.M.,University of Edinburgh | Shehatou G.S.G.,University of Leicester | Shehatou G.S.G.,Mansoura University | And 7 more authors.
Molecular Pharmacology | Year: 2014

Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Storaska A.J.,University of Michigan | Mei J.P.,University of Michigan | Wu M.,Johns Hopkins University | Wu M.,University of Iowa | And 17 more authors.
Cellular Signalling | Year: 2013

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100μM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies. © 2013 Elsevier Inc.

Thirunavukkarasu M.,University of Connecticut Health Center | Selvaraju V.,University of Connecticut Health Center | Dunna N.R.,University of Connecticut Health Center | Foye J.L.C.,Essen BioScience | And 3 more authors.
International Journal of Cardiology | Year: 2013

Background Statins (HMG-CoA reductase inhibitors), are known to improve cardiac function in diabetes-induced cardiovascular disease. We investigated the mechanism by which statins ameliorate cardiac function after myocardial infarction (MI). Simvastatin (S) increased tube formation and migration of HUVEC in vitro. We examined the role of simvastatin on cardiac function in streptozotocin (STZ) induced diabetic rats subjected to MI. Methods Rats were randomly assigned to 1) Control (non-diabetic) Sham (CS); 2) Control (non-diabetic) MI (CMI); 3) Control Statin treated Sham (CSS); 4) Control Statin treated MI (CSMI); 5) Diabetic Sham (DS); 6) Diabetic MI (DMI); 7) Diabetic Statin treated Sham (DSS); 8) Diabetic Statin treated MI (DSMI). Two weeks after STZ/saline injection Simvastatin (1 mg/kg.b.wt) was gavaged for 15 days (d). MI was induced 30 d after treatment by permanent LAD ligation. Results The S treated MI groups exhibited increased arteriolar density (23 ± 0.6 vs. 14.8 ± 0.4 counts/mm2, DSMI vs. DMI) and reduced fibrosis at 30 d post-MI. VEGF measurement by ELISA after 4 d post-MI showed increased expression in DSMI group compared to DMI group. Western blot analysis showed decreased Prolyl-4-Hydroxylase 3 (PHD-3) in DSMI group as compared to DMI group. Echocardiographic analysis 4 weeks after post-MI showed significant improvement in ejection fraction (50.11 ± 1.83 vs. 32.46 ± 2.19%; DSMI vs. DMI) and fractional shortening (26.77 ± 1.12 vs.16.36 ± 1.22%; DSMI vs. DMI) in both statin-treated MI groups regardless of diabetic status. Conclusion These results suggest that statin therapy mitigates impairment of angiogenesis and myocardial dysfunction following MI in the diabetic rat through PHD3 inhibition. © 2013 Elsevier Ireland Ltd. All rights reserved.

Apparatus and methods to improve the Boyden chamber used in cellular biological measurements, allowing quantitative optical microscopy of biological cells in situ without using fluorescent probes or optical staining. A thin, porous membrane separating top and bottom reservoirs includes an array of precisely positioned micropores pores manufactured using a laser-based photo-machining (ablation) process. The membrane may be composed of polyethylene terephthalate (PET), polycarbonate, polyimide, polyether ether ketone (PEEK), polystyrene, or other appropriate material. The pores formed in the membrane may have diameters in the range of 1 to 15 microns and spaced apart at a distance ranging from 10 to 500 microns. A plurality of upper and lower reservoirs may be provided to form a multi-well plate. Potential biological applications where Boyden chamber geometries are currently used include co-culture studies, tissue remodeling studies, cell polarity determinations, endocrine signaling, cell transport, cell permeability, cell invasion and chemotaxis assays.

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