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Yang H.,North Carolina State University | Gurgel P.V.,North Carolina State University | Williams Jr. D.K.,North Carolina State University | Bobay B.G.,North Carolina State University | And 4 more authors.
Journal of Molecular Recognition | Year: 2010

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383-Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. © 2010 John Wiley & Sons, Ltd.

Barth B.M.,University of Alaska Fairbanks | Barth B.M.,Pennsylvania State University | Gustafson S.J.,University of Alaska Fairbanks | Gustafson S.J.,North Carolina Research Campus | And 5 more authors.
Cellular Signalling | Year: 2012

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNFα exposure dramatically increased Mg2+-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNFα exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNFα. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation. © 2011 Elsevier Inc..

Davis M.F.,North Carolina State University | Davis M.F.,University of North Carolina at Chapel Hill | Bobay B.G.,North Carolina State University | Bobay B.G.,North Carolina Research Campus | Franzen S.,North Carolina State University
Biochemistry | Year: 2010

Dehaloperoxidase-hemoglobin (DHP A) is a dual function protein found in the terrebellid polychaete Amphitrite ornata. A. ornata is an annelid, which inhabits estuary mudflats with other polychaetes that secrete a range of toxic brominated phenols. DHPA is capable of binding and oxidatively dehalogenating some of these compounds. DHP A possesses the ability to bind halophenols in a distinct, internal distal binding pocket. Since its discovery, the distal binding pocket has been reported as the sole binding location for halophenols; however, data herein suggest a distinction between inhibitor (monohalogenated phenol) and substrate (trihalogenated phenol) binding locations. Backbone 13Cα, 13Cβ, carbonyl 13C, amide 1H, and amide 15N resonance assignments have been made, and various halophenols were titrated into the protein. 1H- 15N HSQC experiments were collected at stoichiometric intervals during each titration, and binding locations specific for mono- and trihalogenated phenols have been identified. Titration of monohalogenated phenol induced primary changes around the distal binding pocket, while introduction of trihalogenated phenols created alterations of the distal histidine and the local area surrounding W120, a structural region that corresponds to a possible dimer interface region recently observed in X-ray crystal structures of DHP A. ©2010 American Chemical Society.

Hobbs C.A.,North Carolina State University | Bobay B.G.,North Carolina State University | Bobay B.G.,North Carolina Research Campus | Thompson R.J.,North Carolina State University | And 2 more authors.
Journal of Molecular Biology | Year: 2010

Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive spore-forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. ComA belongs to the NarL family of proteins, which are characterized by a C-terminal transcriptional activator domain that consists of a bundle of four helices, where the second and third helices (γ8 and γ9) form a helix-turn-helix DNA-binding domain. Using NMR spectroscopy, the high-resolution 3D solution structure of the C-terminal DNA-binding domain of ComA (ComAC) has been determined. In addition, surface plasmon resonance and NMR protein-DNA titration experiments allowed for the analysis of the interaction of ComAC with its target DNA sequences. Combining the solution structure and biochemical data, a model of ComAC bound to the ComA recognition sequences on the srfA promoter has been developed. The model shows that for DNA binding, ComA uses the conserved helix-turn-helix motif present in other NarL family members. However, the model reveals also that ComA might use a slightly different part of the helix-turn-helix motif and there appears to be some associated domain re-orientation. These observations suggest a basis for DNA binding specificity within the NarL family. © 2010 Elsevier Ltd.

Nieman D.C.,Appalachian State University | Nieman D.C.,North Carolina Research Campus | Cialdella-Kam L.,Appalachian State University | Knab A.M.,Appalachian State University | Shanely R.A.,Appalachian State University
Plant Foods for Human Nutrition | Year: 2012

Red pepper spice (RP) and turmeric (TM) are used as flavorings in foods and for medicinal purposes. Utilizing a randomized, doubled-blinded, placebo-controlled, crossover design (2-week washout), 4-week supplementation with RP (1 g/d) or TM (2.8 g/d) was tested for influences on inflammation and oxidative stress in 62 overweight/obese (body mass index ≥ 27 kg/m2) females (40-75 years) with systemic inflammation (C-reactive protein, CRP ≥ 2 mg/l). Overnight, fasted blood samples were collected pre- and post-supplementation, and analyzed for oxidative stress (F2-isoprostanes, oxidized low density lipoprotein), inflammation (CRP and seven inflammatory cytokines), and metabolic profiles using gas chromatography-mass spectrometry with multivariate partial least square discriminant analysis (PLS-DA). Pre- to post-supplementation measures of inflammation and oxidative stress for both RP and TM did not differ when compared to placebo (all interaction effects, P > 0.05), and global metabolic difference scores calculated through PLS-DA were non-significant (both spices, Q2Y < 0.40). These data indicate that 4-week supplementation with RP or TM at culinary levels does not alter oxidative stress or inflammation in overweight/obese females with systemic inflammation, or cause a significant shift in the global metabolic profile. © 2012 Springer Science+Business Media New York.

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