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Golden Triangle, NC, United States

Anderson K.A.,Duke University | Lin F.,NIEHS | Ribar T.J.,Duke University | Stevens R.D.,Duke University | And 3 more authors.
Molecular Endocrinology | Year: 2012

Ca 2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca2+/CaMdependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system-signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2 floxed mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content. © 2012 by The Endocrine Society. Source

Reagan W.J.,Fizer Global Research and Development | Irizarry-Rovira A.,Eli Lilly and Company | Poitout-Belissent F.,Charles River Laboratories | Provencher Bolliger A.,Charles River Laboratories | And 5 more authors.
Toxicologic Pathology | Year: 2011

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed. © 2011 by The Author(s). Source

Moss M.L.,BioZyme Inc. | Rasmussen F.H.,BioZyme Inc. | Nudelman R.,Teva Pharmaceutical Industries | Dempsey P.J.,University of Michigan | Williams J.,NIEHS
Combinatorial Chemistry and High Throughput Screening | Year: 2010

Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxyfluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)-NH 2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)-NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala- Lys(Fam)-NH 2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 ± 20×10 -2 min -1, 20 ± 10×10 -2 min -1, and 10 ± 3×10 -2 min -1. In addition, a peptide based on the 33kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu-Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)-NH 2 was processed as well but with less efficiency. A more selective substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNFalpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC 50 of 2.1 ± 0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an IC 50 of 280 ± 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high throughput screening assays for ADAM9. © 2010 Bentham Science Publishers Ltd. Source

Smith-Roe S.L.,NIEHS | Patel S.S.,Medical University of South Carolina | Zhou Y.,University of North Carolina at Chapel Hill | Simpson D.A.,University of North Carolina at Chapel Hill | And 4 more authors.
Cell Cycle | Year: 2013

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS -TIPIN complex and CLASP IN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS -TIPIN complex and CLASP IN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS , TIPIN or CLASP IN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASP IN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS -TIPIN and CLASP IN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function. © 2013 Landes Bioscience. Source

Sofer T.,Harvard University | Schifano E.D.,University of Connecticut | Hoppin J.A.,NIEHS | Hou L.,Northwestern University | Baccarelli A.A.,Harvard University
Bioinformatics | Year: 2013

Motivation: DNA methylation is a heritable modifiable chemical process that affects gene transcription and is associated with other molecular markers (e.g. gene expression) and biomarkers (e.g. cancer or other diseases). Current technology measures methylation in hundred of thousands, or millions of CpG sites throughout the genome. It is evident that neighboring CpG sites are often highly correlated with each other, and current literature suggests that clusters of adjacent CpG sites are co-regulated. Results: We develop the Adjacent Site Clustering (A-clustering) algorithm to detect sets of neighboring CpG sites that are correlated with each other. To detect methylation regions associated with exposure, we propose an analysis pipeline for high-dimensional methylation data in which CpG sites within regions identified by A-clustering are modeled as multivariate responses to environmental exposure using a generalized estimating equation approach that assumes exposure equally affects all sites in the cluster. We develop a correlation preserving simulation scheme, and study the proposed methodology via simulations. We study the clusters detected by the algorithm on high dimensional dataset of peripheral blood methylation of pesticide applicators. © The Author 2013. Published by Oxford University Press. All rights reserved. Source

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