41 results on '"Bonacci, G."'
Search Results
2. NOVEL ROBOTIC SOLUTIONS TO IMPROVE MAINTENANCE ON CONDITION
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Frisoli, A., Solazzi, M., Loconsole, C., Tattoli, G., Manno, V., Santamato, G., Caposciutti, M., Masini, P., Menci, M., Desideri, R., and Bonacci, G.
- Subjects
FEM ,Maintenance on Condition ,Maintenance, Robotic, Railway, Inspection, Maintenance on Condition, Computer Vision, FEM ,Maintenance ,Railway ,Inspection ,Computer Vision ,Robotic - Published
- 2016
3. Novel electrophilic omega‐3 fatty acid derivatives are produced by activated macrophages
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Groeger, Alison, primary, Schopfer, F. J., additional, Bonacci, G., additional, Khoo, N., additional, and Freeman, B. A., additional
- Published
- 2008
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4. Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction
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S nchez, M. a C., primary, Chiabrando, G. A., additional, Guglielmone, H. A., additional, Bonacci, G. R., additional, Rabinovich, G. A., additional, and Vides, M. A., additional
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- 1998
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5. Immune Control of Tumors by Antigen Presentation Improvement
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Remedi, M.M., Bonacci, G., Vides, M.A., and Donadio, A.C.
- Abstract
Abstract Tumor cells cannot activate T lymphocytes, since they do not usually express major histocompatibility complex (MHC) class II molecules. Thus, tumor antigens can only be presented indirectly to T cells through professional antigen-presenting cells (APC). In our laboratory, we have treated a tumor cell line (Tu1-A) derived from an induced rat mammary sarcoma in order to increase the expression of MHC class I and class II molecules. In our tumor model, the transference of these induced cells into normal rats generated a tumor mass that exhibited a lower tumor growth rate and an earlier regression as compared to those observed in rats inoculated with wild-type Tu1-A cells. This earlier tumor regression was associated with the development of an antigen-specific immune response. 8587% of the rats in both groups rejected the tumor and were alive at day 60 after tumor cell inoculation. However, in rats treated with wild-type cells the rejection was delayed and took place after tumor ulceration. Rats that had rejected tumors were rechallenged with wild-type cells in order to assay the presence of a long-lived antitumor immunity. All the animals were resistant to the second tumor challenge. We conclude that the development of a specific immune response could be achieved by the superexpression of MHC molecules on tumor cells or when tumor ulceration promotes APC to take up necrotic cells and tumor antigens are presented to T lymphocytes.Copyright © 2003 S. Karger AG, Basel- Published
- 2003
6. Stabilization of homogeneous preparations of pregnancy zone protein lyophilized in the presence of saccharose
- Author
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Bonacci, G., Sanchez, M. C., Gonzalez, M., Ceschin, D., Fidelio, G., Vides, M. A., and Chiabrando, G.
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- 2000
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7. Inhibitory effects of human a2-macroglobulin on Trypanosoma cruzi epimastigote proteinases
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Ramos, A., Remedi, M. S., Sanchez, C., Bonacci, G., Vides, M. A., and Chiabrando, G.
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- 1997
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8. Vision-related quality of life and symptom perception change over time in newly-diagnosed primary open angle glaucoma patients
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Riva, I, Legramandi, L, Rulli, E, Konstas, Ag, Katsanos, A, Oddone, F, Weinreb, Rn, Quaranta, L, Varano, L, Carchedi, T, Talarico, S, Parravano, F, Motolese, I, Bagaglia, Sa, Rossi, Gcm, Lateri, S, Bossolesi, L, Carmassi, L, Rolle, T, Piccini, R, Ratiglia, R, Rossi, A, Gandolfi, S, Tagliavini, Ungaro, N, Fossarello, M, Cuccu, A, Zucca, I, Uva, M, Bonacci, E, Cardarella, G, Tognetto, D, Vattovani, O, Vallon, P, Iannacone, F, Fontana, L, Marchi, S, Manni, G, Iannetta, D, Roberti, G, Rossetti, L, Maggiolo, E, Oneta, O, Sborgia, C, Cantatore, F, Mastropasqua, L, Agnifili, L, Campos, E, Gizzi, C, Giannaccare, G, Pucci, Cassamali, M, Costagliola, C, Traverso, C, Scotto, R, Musolino, M, Landi, L, Bagnis, A, Riva I, Legramandi L, Rulli E, Konstas AG, Katsanos A, Oddone F, Weinreb RN, Quaranta L, Italian Study Group on QoL in Glaucoma: L Varano, T Carchedi, S Talarico, F Parravano, I Motolese, SA Bagaglia, GCM Rossi, S Lateri, L Bossolesi, L Carmassi, T Rolle, R Piccini, R Ratiglia, A Rossi, S Gandolfi, V Tagliavini, N Ungaro, M Fossarello, A Cuccu, I Zucca, M Uva, E Bonacci, G Cardarella, D Tognetto, O Vattovani, P Vallon, F Iannacone, L Fontana, S Marchi, GL Manni, D Jannetta, G Roberti, L Rossetti, E Maggiolo, O Oneta, C Sborgia, F Cantatore, L Mastropasqua, L Agnifili, E Campos, C Gizzi, G Giannaccare, V Pucci, M Cassamali, C Costagliola, C Traverso, R Scotto, M Musolino, L Landi, A Bagnis, Riva, Ivano, Legramandi, Lorenzo, Rulli, Eliana, Konstas, Anastasios G, Katsanos, Andrea, Oddone, Francesco, Weinreb, Robert N, Quaranta, Luciano, Varano, L, Carchedi, T, Talarico, S, Parravano, F, Motolese, I, Bagaglia, Sa, Rossi, Gcm, Lateri, S, Bossolesi, L, Carmassi, L, Rolle, T, Piccini, R, Ratiglia, R, Rossi, A, Gandolfi, S, Tagliavini, Ungaro, N, Fossarello, M, Cuccu, A, Zucca, I, Uva, M, Bonacci, E, Cardarella, G, Tognetto, D, Vattovani, O, Vallon, P, Iannacone, F, Fontana, L, Marchi, S, Manni, Gl, Jannetta, D, Roberti, G, Rossetti, L, Maggiolo, E, Oneta, O, Sborgia, C, Cantatore, F, Mastropasqua, L, Agnifili, L, Campos, E, Gizzi, C, Giannaccare, G, Pucci, Cassamali, M, Costagliola, C, Traverso, C, Scotto, R, Musolino, M, Landi, L, and Bagnis, A.
- Subjects
0301 basic medicine ,Change over time ,medicine.medical_specialty ,Aging ,Visual acuity ,Open angle glaucoma ,genetic structures ,Population ,lcsh:Medicine ,Glaucoma ,Diseases ,Italian Study Group on QoL in Glaucoma ,Neurodegenerative ,Eye ,Article ,03 medical and health sciences ,Medical research ,0302 clinical medicine ,Quality of life ,Clinical Research ,Settore MED/30 ,Internal medicine ,medicine ,POAG ,lcsh:Science ,education ,Prospective cohort study ,Author Correction ,Eye Disease and Disorders of Vision ,education.field_of_study ,Multidisciplinary ,business.industry ,lcsh:R ,Neurosciences ,medicine.disease ,primary open angle glaucoma ,humanities ,eye diseases ,030104 developmental biology ,Symptom perception ,glaucoma ,quality of life ,lcsh:Q ,sense organs ,medicine.symptom ,business ,030217 neurology & neurosurgery - Abstract
To evaluate the change over time of vision-related quality of life (QoL) and glaucoma symptoms in a population of newly-diagnosed primary open angle glaucoma (POAG) patients. Multicenter, prospective study. Consecutive newly-diagnosed POAG patients were enrolled and followed-up for one year. Follow-up visits were scheduled at 6 and 12 months from baseline. At each visit, vision-related QoL and glaucoma-related symptoms were assessed by the means of the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25) and the Glaucoma Symptom Scale (GSS), respectively. Trends over time for NEI-VFQ-25 and GSS scores were evaluated with longitudinal linear mixed models. One-hundred seventy-eight patients were included in the analysis. At baseline, early to moderate glaucoma stages were associated with higher scores for most GSS and NEI-VFQ-25 items, while lower best-corrected visual acuity was associated with lower scores for 4 of the 12 NEI-VFQ-25 items. During the follow-up, all the GSS scores, the NEI-VFQ-25 total score, and 7 of the 12 NEI-VFQ-25 scores significantly improved (p
- Published
- 2019
9. Influence of Sociodemographic Factors on Disease Characteristics and Vision-related Quality of Life in Primary Open-angle Glaucoma Patients: The Italian Primary Open Angle Glaucoma Study (IPOAGS)
- Author
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Riva, Ivano, Legramandi, Lorenzo, Katsanos, Andreas, Oddone, Francesco, Rulli, Eliana, Roberti, Gloria, Quaranta Luciano, Varano L, Carchedi T, Talarico S, Parravano F, Motolese I, Bagaglia SA, Rossi GCM, Lateri S, Bossolesi L, Carmassi L, Rolle T, Piccini R, Ratiglia R, Rossi A, Gandolfi S, Tagliavini V, Ungaro N, Fossarello M, Cucca A, Zucca I, Uva M, Bonacci E, Cardarella G, Tognetto D, Vattovani O, Vallon P, Iannacone F, Fontana L, Marchi S, Manni GL, Jannetta D, Roberti G, Rossetti L, Maggiolo E, Oneta O, Sborgia C, Cantatore F, Mastropasqua L, Agnifili L, Campos E, Gizzi C, Giannaccare G, Pucci V, Cassamali M, Costagliola C, Scotto R, Musolino M, Landi L, Bagnis A., I Riva, L Legramandi, A Katsanos, F Oddone, E Rulli, G Roberti, L Quaranta, Italian Study Group on QoL in Glaucoma: L Varano, T Carchedi, S Talarico, F Parravano, I Motolese, SA Bagaglia, GCM Rossi, S Lateri, L Bossolesi, L Carmassi, T Rolle, R Piccini, R Ratiglia, A Rossi, S Gandolfi, V Tagliavini, N Ungaro, M Fossarello, A Cucca, I Zucca, M Uva, E Bonacci, G Cardarella, D Tognetto, O Vattovani, P Vallon, F Iannacone, L Fontana, S Marchi, GL Manni, D Jannetta, L Rossetti, E Maggiolo, O Oneta, C Sborgia, F Cantatore, L Mastropasqua, L Agnifili, E Campos, C Gizzi, G Giannaccare, V Pucci, M Cassamali, C Costagliola, C Traverso, R Scotto, M Musolino, L Landi, A Bagnis, Riva, Ivano, Legramandi, Lorenzo, Katsanos, Andrea, Oddone, Francesco, Rulli, Eliana, Roberti, Gloria, Quaranta, Luciano, Varano, L, Carchedi, T, Talarico, S, Parravano, F, Motolese, I, Bagaglia, Sa, Rossi, Gcm, Lateri, S, Bossolesi, L, Carmassi, L, Rolle, T, Piccini, R, Ratiglia, R, Rossi, A, Gandolfi, S, Tagliavini, V, Ungaro, N, Fossarello, M, Cucca, A, Zucca, I, Uva, M, Bonacci, E, Cardarella, G, Tognetto, D, Vattovani, O, Vallon, P, Iannacone, F, Fontana, L, Marchi, S, Manni, Gl, Jannetta, D, Roberti, G, Rossetti, L, Maggiolo, E, Oneta, O, Sborgia, C, Cantatore, F, Mastropasqua, L, Agnifili, L, Campos, E, Gizzi, C, Giannaccare, G, Pucci, V, Cassamali, M, Costagliola, C, Scotto, R, Musolino, M, Landi, L, and Bagnis, A.
- Subjects
Male ,medicine.medical_specialty ,Multivariate statistics ,Visual acuity ,Open angle glaucoma ,genetic structures ,Cross-sectional study ,sociodemographic factors ,Visual Acuity ,Glaucoma ,03 medical and health sciences ,0302 clinical medicine ,Quality of life ,Internal medicine ,Settore MED/30 ,Sickness Impact Profile ,Surveys and Questionnaires ,medicine ,Humans ,030212 general & internal medicine ,Intraocular Pressure ,Vision, Ocular ,Aged ,Demography ,business.industry ,Middle Aged ,medicine.disease ,eye diseases ,glaucoma ,quality of life ,Ophthalmology ,Cross-Sectional Studies ,Italy ,sociodemographic factor ,030221 ophthalmology & optometry ,Quality of Life ,Marital status ,Disease characteristics ,Female ,medicine.symptom ,business ,Glaucoma, Open-Angle - Abstract
Purpose: The purpose of this article was to evaluate the potential association between sociodemographic factors with clinical characteristics, vision-related quality of life (QoL), and glaucoma-related symptoms scores in a large cohort of primary open-angle glaucoma patients. Materials and Methods: Multicenter, cross-sectional study involving academic and nonacademic centers. Previously diagnosed primary open-angle glaucoma patients aged >18 years were enrolled. At baseline, information on demographic characteristics, social, medical and ocular history, clinical presentation and treatments was collected. Vision-related QoL was evaluated by means of the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25), while glaucoma-related symptoms were evaluated using the Glaucoma Symptom Scale (GSS) questionnaire. The associations between sociodemographic factors with clinical characteristics (mean deviation, pattern standard deviation, best-corrected visual acuity), NEI-VFQ-25, and GSS scores were evaluated by means of univariate and multivariate general linear models. Results: A total of 3227 patients were enrolled. Older age and male sex were significantly associated with lower mean deviation (P
- Published
- 2018
10. What do primary care providers want to know when caring for patients living with frailty? An analysis of eConsult communications between primary care providers and specialists.
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Karunananthan S, Bonacci G, Fung C, Huang A, Robert B, McCutcheon T, Houghton D, Hakimjavadi R, Keely E, and Liddy C
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- Humans, Cross-Sectional Studies, Health Services Accessibility, Ontario, Primary Health Care methods, Referral and Consultation, Aged, Frailty diagnosis, Frailty therapy, Remote Consultation
- Abstract
Background: Frailty is a complex condition that primary care providers (PCPs) are managing in increasing numbers, yet there is no clear guidance or training for frailty care., Objectives: The present study examined eConsult questions PCPs asked specialists about patients with frailty, the specialists' responses, and the impact of eConsult on the care of these patients., Design: Cross-sectional observational study., Setting: ChamplainBASE™ eConsult located in Eastern Ontario, Canada., Participants: Sixty one eConsult cases closed by PCPs in 2019 that use the terms "frail" or "frailty" to describe patients 65 years of age or older., Measurements: The Taxonomy of Generic Clinical Questions (TGCQ) was used to classify PCP questions and the International Classification for Primary Care 3 (ICPC-3) was used to classify the clinical content of each eConsult. The impact of eConsult on patient care was measured by PCP responses to a mandatory survey., Results: PCPs most frequently directed their questions to cardiology (n = 7; 11%), gastroenterology (n = 7; 11%), and endocrinology (n = 6; 10%). Specialist answers most often pertained to medications (n = 63, 46%), recommendations for clinical investigation (n = 24, 17%), and diagnoses (n = 22, 16%). Specialist responses resulted in PCPs avoiding referral in 57% (n = 35) of cases whereas referrals were still required in 15% (n = 9) of cases. Specialists responded to eConsults in a median 1.11 days (IQR = 0.3-4.7), and 95% (n = 58) of cases received a response within 7 days. Specialists recorded a median of 15 min to respond (IQR = 10-20), with a median cost of $50.00 CAD (IQR = 33.33 - 66.66) per eConsult., Conclusions: Through the analysis of questions and responses submitted to eConsult, this study provides novel information on PCP knowledge gaps and approaches to care for patients living with frailty. Furthermore, these analyses provide evidence that eConsult is a feasible and valuable tool for improving care for patients with frailty in primary care settings., (© 2024. The Author(s).)
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- 2024
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11. Protective Effect of NO 2 -OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.
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Vaglienti MV, Subirada PV, Joray MB, Bonacci G, and Sánchez MC
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- Humans, Ependymoglial Cells metabolism, Gliosis metabolism, Oxidative Stress, Hypoxia metabolism, Inflammation metabolism, RNA, Messenger metabolism, Nitrogen Dioxide metabolism, Nitrogen Dioxide pharmacology, Vascular Endothelial Growth Factor A metabolism
- Abstract
Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α
2 -macroglobulin (α2 M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2 -OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2 -OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2 -OA prevented the ROS increase and reduced the gliosis induced by α2 M. Finally, when hypoxic MGCs were incubated with NO2 -OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1β), NO2 -OA significantly reduced VEGF mRNA levels. Furthermore, NO2 -OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2 -OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.- Published
- 2023
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12. Quantification of Reactive Oxygen Species Using 2',7'-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells.
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Vaglienti MV, Subirada PV, Barcelona PF, Bonacci G, and Sanchez MC
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- Antioxidants metabolism, Antioxidants pharmacology, Flow Cytometry, Fluoresceins, Humans, Infant, Newborn, Kelch-Like ECH-Associated Protein 1 metabolism, Reactive Oxygen Species metabolism, Ependymoglial Cells, NF-E2-Related Factor 2 metabolism
- Abstract
The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.
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- 2022
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13. Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation.
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Carranza ADV, Bonacci G, Moran Y, Asprelli P, Carrari F, and Asis R
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- Animals, Caenorhabditis elegans genetics, DNA-Binding Proteins, Dietary Supplements, NF-E2-Related Factor 2 genetics, Tandem Mass Spectrometry, Transcription Factors, Caenorhabditis elegans Proteins, Solanum lycopersicum
- Abstract
Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, β- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
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- 2022
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14. Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages.
- Author
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Vazquez MM, Gutierrez MV, Salvatore SR, Puiatti M, Dato VA, Chiabrando GA, Freeman BA, Schopfer FJ, and Bonacci G
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- Animals, Cholesterol, Foam Cells metabolism, Ligands, Lipoproteins, LDL metabolism, Macrophages metabolism, Mice, CD36 Antigens genetics, Oleic Acid
- Abstract
Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO
2 -OA) on macrophage lipid metabolism. Pure synthetic NO2 -OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2 -OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2 -OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2 -OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2 -OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2020
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15. LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells.
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Actis Dato V, Benitez-Amaro A, de Gonzalo-Calvo D, Vazquez M, Bonacci G, Llorente-Cortés V, and Chiabrando GA
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- Animals, Cell Line, Endocytosis, Mice, Protein Aggregates, Cholesterol Esters metabolism, Insulin metabolism, Lipoproteins, LDL metabolism, Low Density Lipoprotein Receptor-Related Protein-1 metabolism
- Abstract
A bstract: The cardiovascular disease (CVD) frequently developed during metabolic syndrome and type-2 diabetes mellitus is associated with increased levels of aggregation-prone small LDL particles. Aggregated LDL (aggLDL) internalization is mediated by low-density lipoprotein receptor-related protein-1 (LRP1) promoting intracellular cholesteryl ester (CE) accumulation. Additionally, LRP1 plays a key function in the regulation of insulin receptor (IR) and glucose transporter type 4 (GLUT4) activities. Nevertheless, the link between LRP1, CE accumulation, and insulin response has not been previously studied in cardiomyocytes. We aimed to identify mechanisms through which aggLDL, by its interaction with LRP1, produce CE accumulation and affects the insulin-induced intracellular signaling and GLUT4 trafficking in HL-1 cells. We demonstrated that LRP1 mediates the endocytosis of aggLDL and promotes CE accumulation in these cells. Moreover, aggLDL reduced the molecular association between IR and LRP1 and impaired insulin-induced intracellular signaling activation. Finally, aggLDL affected GLUT4 translocation to the plasma membrane and the 2-NBDG uptake in insulin-stimulated cells. We conclude that LRP1 is a key regulator of the insulin response, which can be altered by CE accumulation through LRP1-mediated aggLDL endocytosis., Competing Interests: The authors declare no conflicts of interest.
- Published
- 2020
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16. Nitro-fatty acid formation and metabolism.
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Buchan GJ, Bonacci G, Fazzari M, Salvatore SR, and Gelhaus Wendell S
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- Animals, Humans, Nitric Oxide metabolism, Oxidation-Reduction, Fatty Acids biosynthesis, Fatty Acids metabolism, Nitro Compounds metabolism
- Abstract
Nitro-fatty acids (NO
2 -FA) are pleiotropic modulators of redox signaling pathways. Their effects on inflammatory signaling have been studied in great detail in cell, animal and clinical models primarily using exogenously administered nitro-oleic acid. While we know a considerable amount regarding NO2 -FA signaling, endogenous formation and metabolism is relatively unexplored. This review will cover what is currently known regarding the proposed mechanisms of NO2 -FA formation, dietary modulation of endogenous NO2 -FA levels, pathways of NO2 -FA metabolism and the detection of NO2 -FA and corresponding metabolites., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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17. New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases.
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Sanmarco LM, Eberhardt N, Ponce NE, Cano RC, Bonacci G, and Aoki MP
- Abstract
Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with Trypanosoma cruzi , the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies.
- Published
- 2018
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18. Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.
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Müller V, Bonacci G, Batthyany C, Amé MV, Carrari F, Gieco J, and Asis R
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- Aflatoxins metabolism, Arachis immunology, Arachis microbiology, Gene Expression Regulation, Plant, Plant Diseases microbiology, Plant Proteins genetics, Plant Proteins isolation & purification, Plant Proteins metabolism, Protease Inhibitors isolation & purification, Seeds genetics, Seeds immunology, Seeds microbiology, Arachis genetics, Aspergillus metabolism, Plant Diseases immunology, Protease Inhibitors metabolism
- Abstract
Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.
- Published
- 2017
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19. Fatty acid nitroalkenes induce resistance to ischemic cardiac injury by modulating mitochondrial respiration at complex II.
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Koenitzer JR, Bonacci G, Woodcock SR, Chen CS, Cantu-Medellin N, Kelley EE, and Schopfer FJ
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- Alkenes pharmacology, Animals, Cardiotonic Agents metabolism, Cardiotonic Agents pharmacology, Cell Line, Electron Transport Complex II antagonists & inhibitors, Electron Transport Complex III metabolism, Fatty Acids pharmacology, Hydrogen-Ion Concentration, Male, Myoblasts, Cardiac metabolism, Myocardial Reperfusion Injury metabolism, Nitric Oxide metabolism, Nitro Compounds pharmacology, Oxidation-Reduction, Rats, Superoxides metabolism, Alkenes metabolism, Cell Respiration, Electron Transport Complex II metabolism, Fatty Acids metabolism, Mitochondria, Heart metabolism, Myocardial Ischemia metabolism, Nitro Compounds metabolism
- Abstract
Nitro-fatty acids (NO2-FA) are metabolic and inflammatory-derived electrophiles that mediate pleiotropic signaling actions. It was hypothesized that NO2-FA would impact mitochondrial redox reactions to induce tissue-protective metabolic shifts in cells. Nitro-oleic acid (OA-NO2) reversibly inhibited complex II-linked respiration in isolated rat heart mitochondria in a pH-dependent manner and suppressed superoxide formation. Nitroalkylation of Fp subunit was determined by BME capture and the site of modification by OA-NO2 defined by mass spectrometric analysis. These effects translated into reduced basal and maximal respiration and favored glycolytic metabolism in H9C2 cardiomyoblasts as assessed by extracellular H(+) and O2 flux analysis. The perfusion of NO2-FA induced acute cardioprotection in an isolated perfused heart ischemia/reperfusion (IR) model as evidenced by significantly higher rate-pressure products. Together these findings indicate that NO2-FA can promote cardioprotection by inducing a shift from respiration to glycolysis and suppressing reactive species formation in the post-ischemic interval., (Copyright © 2015. Published by Elsevier B.V.)
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- 2016
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20. Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity.
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Kelley EE, Baust J, Bonacci G, Golin-Bisello F, Devlin JE, St Croix CM, Watkins SC, Gor S, Cantu-Medellin N, Weidert ER, Frisbee JC, Gladwin MT, Champion HC, Freeman BA, and Khoo NK
- Subjects
- Adipose Tissue metabolism, Animals, Body Weight physiology, Hypertension, Pulmonary complications, Insulin metabolism, Insulin Resistance physiology, Male, Mice, Mice, Inbred C57BL, Obesity complications, Blood Glucose metabolism, Diet, High-Fat adverse effects, Fatty Acids metabolism, Glucose Intolerance metabolism, Hypertension, Pulmonary metabolism, Obesity metabolism
- Abstract
Aims: Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions., Methods and Results: It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels., Conclusions: These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.
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- 2014
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21. Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes.
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Woodcock SR, Salvatore SR, Bonacci G, Schopfer FJ, and Freeman BA
- Subjects
- Biomimetics, Chromatography, High Pressure Liquid, Magnetic Resonance Spectroscopy, Tandem Mass Spectrometry, Linoleic Acids, Conjugated chemistry, Lipids chemistry, Nitro Compounds chemistry
- Abstract
Nitro-conjugated linoleic acids (NO2-cLA), endogenous nitrodiene lipids which act as inflammatory signaling mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid (CLA). Structures were elucidated by means of detailed NMR and HPLC-MS/MS spectroscopic analysis and the relative double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif with few biological examples extant.
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- 2014
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22. Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.
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Vitturi DA, Chen CS, Woodcock SR, Salvatore SR, Bonacci G, Koenitzer JR, Stewart NA, Wakabayashi N, Kensler TW, Freeman BA, and Schopfer FJ
- Subjects
- Alcohol Oxidoreductases genetics, Animals, Heme Oxygenase-1 genetics, Heme Oxygenase-1 metabolism, Hep G2 Cells, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Kelch-Like ECH-Associated Protein 1, NF-E2-Related Factor 2 genetics, NF-E2-Related Factor 2 metabolism, Oleic Acid genetics, Rats, Alcohol Oxidoreductases metabolism, Liver metabolism, Nitro Compounds metabolism, Oleic Acid metabolism, Signal Transduction physiology, Stearic Acids metabolism
- Abstract
Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.
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- 2013
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23. Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine.
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Salvatore SR, Vitturi DA, Baker PR, Bonacci G, Koenitzer JR, Woodcock SR, Freeman BA, and Schopfer FJ
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- Fatty Acids chemistry, Fatty Acids metabolism, Healthy Volunteers, Humans, Molecular Structure, Nitro Compounds chemistry, Nitro Compounds metabolism, Fatty Acids urine, Nitro Compounds urine
- Abstract
The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.
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- 2013
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24. Nitrated fatty acids: synthesis and measurement.
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Woodcock SR, Bonacci G, Gelhaus SL, and Schopfer FJ
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- Fatty Acids analysis, Fatty Acids chemical synthesis, Nitrates analysis, Nitrates chemical synthesis
- Abstract
Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography-mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2013
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25. Conjugated linoleic acid is a preferential substrate for fatty acid nitration.
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Bonacci G, Baker PR, Salvatore SR, Shores D, Khoo NK, Koenitzer JR, Vitturi DA, Woodcock SR, Golin-Bisello F, Cole MP, Watkins S, St Croix C, Batthyany CI, Freeman BA, and Schopfer FJ
- Subjects
- Animals, Cell Line, Humans, Mice, Mice, Inbred C57BL, Mitochondria metabolism, Nitric Oxide metabolism, Signal Transduction, Fatty Acids metabolism, Linoleic Acid metabolism, Nitrates metabolism, Nitrites metabolism
- Abstract
The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10(5) greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO(2)-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.
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- 2012
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26. The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization.
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Bonacci G, Fletcher J, Devani M, Dwivedi H, Keller R, and Chang C
- Subjects
- Animals, Arginine genetics, Arginine metabolism, Cadherins metabolism, Cell Adhesion, Cell Lineage, Cell Movement, Embryo, Nonmammalian cytology, Humans, Phosphorylation, Protein Binding, Protein-Tyrosine Kinases genetics, Xenopus laevis genetics, Actins metabolism, Cytoplasm enzymology, Embryo, Nonmammalian enzymology, Gastrulation, Protein-Tyrosine Kinases metabolism, Xenopus laevis embryology, Xenopus laevis metabolism
- Abstract
Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation., (© 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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27. Gas-phase fragmentation analysis of nitro-fatty acids.
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Bonacci G, Asciutto EK, Woodcock SR, Salvatore SR, Freeman BA, and Schopfer FJ
- Subjects
- Gases chemistry, Linoleic Acids chemistry, Nitriles chemistry, Nitrites chemistry, Fatty Acids chemistry, Nitro Compounds chemistry, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
Nitro-fatty acids are electrophilic signaling mediators formed in increased amounts during inflammation by nitric oxide and nitrite-dependent redox reactions. A more rigorous characterization of endogenously-generated species requires additional understanding of their gas-phase induced fragmentation. Thus, collision induced dissociation (CID) of nitroalkane and nitroalkene groups in fatty acids were studied in the negative ion mode to provide mass spectrometric tools for their structural characterization. Fragmentation of nitroalkanes occurred mainly through loss of the NO(2)(-) anion or neutral loss of HNO(2). The CID of nitroalkenes proceeds via a more complex cyclization, followed by fragmentation to nitrile and aldehyde products. Gas-phase fragmentation of nitroalkene functional groups with additional γ or δ unsaturation occurred through a multiple step cyclization reaction process, leading to 5 and 6 member ring heterocyclic products and carbon chain fragmentation. Cyclization products were not obtained during nitroalkane fragmentation, highlighting the role of double bond π electrons during NO(2)(-) rearrangements, stabilization and heterocycle formation. The proposed structures, mechanisms and products of fragmentation are supported by analysis of (13)C and (15)N labeled parent molecules, 6 different nitroalkene positional isomers, 6 nitroalkane positional isomers, accurate mass determinations at high resolution and quantum mechanics calculations. Multiple key diagnostic ion fragments were obtained through this analysis, allowing for the precise placement of double bonds and sites of fatty acid nitration, thus supporting an ability to predict nitro positions in biological samples.
- Published
- 2011
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28. Electrophilic fatty acids regulate matrix metalloproteinase activity and expression.
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Bonacci G, Schopfer FJ, Batthyany CI, Rudolph TK, Rudolph V, Khoo NK, Kelley EE, and Freeman BA
- Subjects
- Animals, Carcinogens pharmacology, Cell Line, Enzyme Activation drug effects, Enzyme Precursors genetics, Humans, Hypoglycemic Agents pharmacology, Inflammation enzymology, Inflammation genetics, Inflammation pathology, Matrix Metalloproteinase 9 genetics, Metalloendopeptidases genetics, Mice, Mice, Knockout, Oleic Acids metabolism, PPAR gamma antagonists & inhibitors, PPAR gamma genetics, PPAR gamma metabolism, Protein Structure, Tertiary, Rosiglitazone, Tetradecanoylphorbol Acetate pharmacology, Thiazolidinediones pharmacology, Transcription, Genetic drug effects, Transcription, Genetic genetics, Enzyme Precursors biosynthesis, Gene Expression Regulation, Enzymologic, Matrix Metalloproteinase 9 biosynthesis, Metalloendopeptidases biosynthesis, Oleic Acids pharmacology
- Abstract
Nitro-fatty acids (NO(2)-FA) are electrophilic signaling mediators formed by reactions of nitric oxide and nitrite. NO(2)-FA exert anti-inflammatory signaling actions through post-translational protein modifications. We report that nitro-oleic acid (OA-NO(2)) stimulates proMMP-7 and proMMP-9 proteolytic activity via adduction of the conserved cysteine switch domain thiolate. Biotin-labeled OA-NO(2) showed this adduction occurs preferentially with latent forms of MMP, confirming a role for thiol alkylation by OA-NO(2) in MMP activation. In addition to regulating pro-MMP activation, MMP expression was modulated by OA-NO(2) via activation of peroxisome proliferator-activated receptor-γ. MMP-9 transcription was decreased in phorbol 12-myristate 13-acetate-stimulated THP-1 macrophages to an extent similar to that induced by the peroxisome proliferator-activated receptor-γ agonist Rosiglitazone. This was affirmed using a murine model of atherosclerosis, ApoE(-/-) mice, where in vivo OA-NO(2) administration suppressed MMP expression in atherosclerotic lesions. These findings reveal that electrophilic fatty acid derivatives can serve as effectors during inflammation, first by activating pro-MMP proteolytic activity via alkylation of the cysteine switch domain, and then by transcriptionally inhibiting MMP expression, thereby limiting the further progression of inflammatory processes.
- Published
- 2011
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29. Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism.
- Author
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Kansanen E, Bonacci G, Schopfer FJ, Kuosmanen SM, Tong KI, Leinonen H, Woodcock SR, Yamamoto M, Carlberg C, Ylä-Herttuala S, Freeman BA, and Levonen AL
- Subjects
- Animals, Chromatography, Liquid methods, Gene Expression Regulation, Humans, Kelch-Like ECH-Associated Protein 1, Linoleic Acids chemistry, Mass Spectrometry methods, Mice, Mutation, Nitro Compounds chemistry, Oleic Acids chemistry, Protein Processing, Post-Translational, Signal Transduction, Transcription Factors metabolism, Ubiquitin-Protein Ligases chemistry, Adaptor Proteins, Signal Transducing chemistry, Cysteine chemistry, Cytoskeletal Proteins chemistry, Fatty Acids chemistry, Intracellular Signaling Peptides and Proteins chemistry, NF-E2-Related Factor 2 chemistry
- Abstract
Nitro-fatty acids (NO(2)-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO(2)(-))-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO(2)) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO(2)-reactive Cys residues in Keap1 were Cys(38), Cys(226), Cys(257), Cys(273), Cys(288), and Cys(489). Of these, Cys(273) and Cys(288) accounted for ∼50% of OA-NO(2) reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO(2)-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO(2) reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO(2) enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO(2) can therefore be categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.
- Published
- 2011
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30. Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids.
- Author
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Groeger AL, Cipollina C, Cole MP, Woodcock SR, Bonacci G, Rudolph TK, Rudolph V, Freeman BA, and Schopfer FJ
- Subjects
- Anti-Inflammatory Agents metabolism, Anti-Inflammatory Agents pharmacology, Borohydrides pharmacology, Cell Line, Cell Membrane metabolism, Docosahexaenoic Acids chemistry, Docosahexaenoic Acids metabolism, Fatty Acids, Unsaturated metabolism, Fatty Acids, Unsaturated pharmacology, Glutathione metabolism, Humans, Hydroxylation, Interleukin-10 genetics, Interleukin-6 genetics, Macrophages drug effects, Macrophages metabolism, PPAR gamma metabolism, PPAR gamma pharmacology, Anti-Inflammatory Agents chemical synthesis, Cyclooxygenase 2 metabolism, Fatty Acids, Omega-3 metabolism
- Abstract
Electrophilic fatty acids are generated during inflammation by non-enzymatic reactions and can modulate inflammatory responses. We used a new mass spectrometry-based electrophile capture strategy to reveal the formation of electrophilic oxo-derivatives (EFOX) from the omega-3 fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These EFOX were generated by a cyclooxygenase-2 (COX-2)-catalyzed mechanism in activated macrophages. Modulation of COX-2 activity by aspirin increased the rate of EFOX production and their intracellular levels. Owing to their electrophilic nature, EFOX adducted to cysteine and histidine residues of proteins and activated Nrf2-dependent anti-oxidant gene expression. We confirmed the anti-inflammatory nature of DHA- and DPA-derived EFOX by showing that they can act as peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and inhibit pro-inflammatory cytokine and nitric oxide production, all within biological concentration ranges. These data support the idea that EFOX are signaling mediators that transduce the beneficial clinical effects of omega-3 fatty acids, COX-2 and aspirin.
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- 2010
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31. Nitro-fatty acids reduce atherosclerosis in apolipoprotein E-deficient mice.
- Author
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Rudolph TK, Rudolph V, Edreira MM, Cole MP, Bonacci G, Schopfer FJ, Woodcock SR, Franek A, Pekarova M, Khoo NK, Hasty AH, Baldus S, and Freeman BA
- Subjects
- Actins metabolism, Animals, Anti-Inflammatory Agents administration & dosage, Antioxidants administration & dosage, Aortic Diseases genetics, Aortic Diseases metabolism, Aortic Diseases pathology, Apolipoproteins E genetics, Atherosclerosis genetics, Atherosclerosis metabolism, Atherosclerosis pathology, Cell Adhesion Molecules metabolism, Cells, Cultured, Chemokine CCL2 metabolism, Collagen metabolism, Disease Models, Animal, Dose-Response Relationship, Drug, Foam Cells drug effects, Foam Cells metabolism, Injections, Subcutaneous, Lipoproteins, LDL metabolism, Male, Mice, Mice, Knockout, Oleic Acids administration & dosage, Oxidants metabolism, Oxidative Stress drug effects, Phosphorylation, STAT1 Transcription Factor metabolism, Signal Transduction drug effects, Anti-Inflammatory Agents pharmacology, Antioxidants pharmacology, Aortic Diseases prevention & control, Apolipoproteins E deficiency, Atherosclerosis prevention & control, Oleic Acids pharmacology
- Abstract
Objective: Inflammatory processes and foam cell formation are key determinants in the initiation and progression of atherosclerosis. Electrophilic nitro-fatty acids, byproducts of nitric oxide- and nitrite-dependent redox reactions of unsaturated fatty acids, exhibit antiinflammatory signaling actions in inflammatory and vascular cell model systems. The in vivo action of nitro-fatty acids in chronic inflammatory processes such as atherosclerosis remains to be elucidated., Methods and Results: Herein, we demonstrate that subcutaneously administered 9- and 10-nitro-octadecenoic acid (nitro-oleic acid) potently reduced atherosclerotic lesion formation in apolipoprotein E-deficient mice. Nitro-fatty acids did not modulate serum lipoprotein profiles. Immunostaining and gene expression analyses revealed that nitro-oleic acid attenuated lesion formation by suppressing tissue oxidant generation, inhibiting adhesion molecule expression, and decreasing vessel wall infiltration of inflammatory cells. In addition, nitro-oleic acid reduced foam cell formation by attenuating oxidized low-density lipoprotein-induced phosphorylation of signal transducer and activator of transcription-1, a transcription factor linked to foam cell formation in atherosclerotic plaques. Atherosclerotic lesions of nitro-oleic acid-treated animals also showed an increased content of collagen and alpha-smooth muscle actin, suggesting conferral of higher plaque stability., Conclusion: These results reveal the antiatherogenic actions of electrophilic nitro-fatty acids in a murine model of atherosclerosis.
- Published
- 2010
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32. Covalent peroxisome proliferator-activated receptor gamma adduction by nitro-fatty acids: selective ligand activity and anti-diabetic signaling actions.
- Author
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Schopfer FJ, Cole MP, Groeger AL, Chen CS, Khoo NK, Woodcock SR, Golin-Bisello F, Motanya UN, Li Y, Zhang J, Garcia-Barrio MT, Rudolph TK, Rudolph V, Bonacci G, Baker PR, Xu HE, Batthyany CI, Chen YE, Hallis TM, and Freeman BA
- Subjects
- 3T3-L1 Cells, Amino Acid Sequence, Amino Acid Substitution, Animals, Base Sequence, Blood Glucose metabolism, Cell Line, DNA Primers metabolism, Diabetes Mellitus drug therapy, Diabetes Mellitus metabolism, Fatty Acids, Unsaturated chemistry, Humans, Hypoglycemic Agents chemistry, In Vitro Techniques, Insulin blood, Ligands, Lipid Metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Obese, Molecular Sequence Data, Mutagenesis, Site-Directed, Nitro Compounds chemistry, Oleic Acid chemistry, Oleic Acid pharmacology, PPAR gamma chemistry, PPAR gamma genetics, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Rosiglitazone, Signal Transduction, Tandem Mass Spectrometry, Thiazolidinediones pharmacology, Fatty Acids, Unsaturated pharmacology, Hypoglycemic Agents pharmacology, Nitro Compounds pharmacology, PPAR gamma agonists, PPAR gamma metabolism
- Abstract
The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.
- Published
- 2010
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33. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion.
- Author
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Rudolph V, Rudolph TK, Schopfer FJ, Bonacci G, Woodcock SR, Cole MP, Baker PR, Ramani R, and Freeman BA
- Subjects
- Alkylation, Animals, Apoptosis, Disease Models, Animal, Echocardiography, Inflammation etiology, Male, Mice, Mice, Inbred C57BL, Myocardial Ischemia pathology, Neutrophil Infiltration, Proline analogs & derivatives, Proline pharmacology, Thiocarbamates pharmacology, Transcription Factor RelA metabolism, Fatty Acids metabolism, Myocardial Ischemia metabolism, Myocardial Reperfusion Injury prevention & control, Nitric Oxide metabolism
- Abstract
Aims: Nitrated fatty acids (NO(2)-FA) have been identified as endogenous anti-inflammatory signalling mediators generated by oxidative inflammatory reactions. Herein the in vivo generation of nitro-oleic acid (OA-NO(2)) and nitro-linoleic acid (LNO(2)) was measured in a murine model of myocardial ischaemia and reperfusion (I/R) and the effect of exogenous administration of OA-NO(2) on I/R injury was evaluated., Methods and Results: In C57/BL6 mice subjected to 30 min of coronary artery ligation, endogenous OA-NO(2) and LNO(2) formation was observed after 30 min of reperfusion, whereas no NO(2)-FA were detected in sham-operated mice and mice with myocardial infarction without reperfusion. Exogenous administration of 20 nmol/g body weight OA-NO(2) during the ischaemic episode induced profound protection against I/R injury with a 46% reduction in infarct size (normalized to area at risk) and a marked preservation of left ventricular function as assessed by transthoracic echocardiography, compared with vehicle-treated mice. Administration of OA-NO(2) inhibited activation of the p65 subunit of nuclear factor kappaB (NFkappaB) in I/R tissue. Experiments using the NFkappaB inhibitor pyrrolidinedithiocarbamate also support that protection lent by OA-NO(2) was in part mediated by inhibition of NFkappaB. OA-NO(2) inhibition of NFkappaB activation was accompanied by suppression of downstream intercellular adhesion molecule 1 and monocyte chemotactic protein 1 expression, neutrophil infiltration, and myocyte apoptosis., Conclusion: This study reveals the de novo generation of fatty acid nitration products in vivo and reveals the anti-inflammatory and potential therapeutic actions of OA-NO(2) in myocardial I/R injury.
- Published
- 2010
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34. Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.
- Author
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Rudolph V, Schopfer FJ, Khoo NK, Rudolph TK, Cole MP, Woodcock SR, Bonacci G, Groeger AL, Golin-Bisello F, Chen CS, Baker PR, and Freeman BA
- Subjects
- Animals, Glutathione metabolism, Humans, Mice, Organ Specificity drug effects, Oxidation-Reduction drug effects, Plasma metabolism, Signal Transduction drug effects, Fatty Acids metabolism, Fatty Acids pharmacology, Liver metabolism, Nitro Compounds metabolism, Nitro Compounds pharmacology, Proteins metabolism
- Abstract
Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and beta-oxidation reactions that terminate at the site of acyl-chain nitration.
- Published
- 2009
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35. Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase.
- Author
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Kelley EE, Batthyany CI, Hundley NJ, Woodcock SR, Bonacci G, Del Rio JM, Schopfer FJ, Lancaster JR Jr, Freeman BA, and Tarpey MM
- Subjects
- Animals, Aorta cytology, Cattle, Dithiothreitol metabolism, Endothelial Cells cytology, Fatty Acids chemistry, Glutathione metabolism, Inhibitory Concentration 50, Mercaptoethanol metabolism, Oxygen chemistry, Signal Transduction, Superoxides metabolism, Enzyme Inhibitors pharmacology, Oleic Acids metabolism, Xanthine Dehydrogenase metabolism
- Abstract
Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.
- Published
- 2008
- Full Text
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36. Bivalirudin decreases NO bioavailability by vascular immobilization of myeloperoxidase.
- Author
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Rudolph V, Rudolph TK, Schopfer FJ, Bonacci G, Lau D, Szöcs K, Klinke A, Meinertz T, Freeman BA, and Baldus S
- Subjects
- Aged, Animals, Biological Availability, Cattle, Female, Fluorescent Antibody Technique, Hirudins metabolism, Humans, Male, Methylation, Middle Aged, Peptide Fragments metabolism, Peroxidase blood, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, Spectrometry, Mass, Electrospray Ionization, Tyrosine analogs & derivatives, Tyrosine metabolism, Vasodilation, Antithrombins pharmacology, Hirudins pharmacology, Nitric Oxide metabolism, Peptide Fragments pharmacology, Peroxidase metabolism
- Abstract
Bivalirudin, a direct thrombin inhibitor, has emerged as an important alternative to heparin in patients undergoing percutaneous coronary intervention. However, it remains elusive if potentially adverse extracoagulant properties are responsible for the fact that its favorable effects in clinical studies are mainly driven by a reduction in bleeding events. The aim of the current study was to determine the effects and mechanisms of acute treatment with bivalirudin in comparison to heparin on NO bioavailability, an important factor for the pathogenesis of ischemic events. In particular, we studied the interaction between bivalirudin and myeloperoxidase (MPO), a leukocyte-derived enzyme that consumes endothelial-derived nitric oxide (NO), modifies a variety of biological targets, and thus affects the integrity of the vessel wall. In patients undergoing elective percutaneous coronary intervention, bivalirudin, in contrast to heparin, exhibited a significant decrease in plasma MPO levels (p = 0.03) accompanied by a deterioration of flow-mediated dilation (p = 0.02), a surrogate for endothelial NO bioavailability. In vitro experiments revealed avid binding of bivalirudin to both bovine aortic endothelial cells (BAEC) and MPO. Methylation of bivalirudin carboxyl groups at the carboxyl-terminal end revealed the specific binding site of bivalirudin to MPO. Bivalirudin-facilitated binding of MPO to BAEC resulted also in functional changes in terms of increased NO consumption as well as enhanced MPO-mediated redox modifications. These results illustrate dichotomous extracoagulant properties of heparins and thrombin inhibitors and suggest that bivalirudin acutely impairs endothelial NO bioavailability, thereby underscoring the potentially critical role of MPO as a mediator of vascular function.
- Published
- 2008
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37. Sparing effects of selenium and ascorbic acid on vitamin C and E in guinea pig tissues.
- Author
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Bertinato J, Hidiroglou N, Peace R, Cockell KA, Trick KD, Jee P, Giroux A, Madère R, Bonacci G, Iskandar M, Hayward S, Giles N, and L'Abbé MR
- Subjects
- Animals, Glutathione blood, Glutathione Peroxidase drug effects, Glutathione Peroxidase metabolism, Guinea Pigs, Kinetics, Liver drug effects, Liver metabolism, Male, Models, Animal, Ascorbic Acid metabolism, Ascorbic Acid pharmacology, Selenium pharmacology, Vitamin E metabolism
- Abstract
Background: Selenium (Se), vitamin C and vitamin E function as antioxidants within the body. In this study, we investigated the effects of reduced dietary Se and L-ascorbic acid (AA) on vitamin C and alpha-tocopherol (AT) status in guinea pig tissues., Methods: Male Hartley guinea pigs were orally dosed with a marginal amount of AA and fed a diet deficient (Se-D/MC), marginal (Se-M/MC) or normal (Se-N/MC) in Se. An additional diet group (Se-N/NC) was fed normal Se and dosed with a normal amount of AA. Guinea pigs were killed after 5 or 12 weeks on the experimental diets at 24 and 48 hours post AA dosing., Results: Liver Se-dependent glutathione peroxidase activity was decreased (P < 0.05) in guinea pigs fed Se or AA restricted diets. Plasma total glutathione concentrations were unaffected (P > 0.05) by reduction in dietary Se or AA. All tissues examined showed a decrease (P < 0.05) in AA content in Se-N/MC compared to Se-N/NC guinea pigs. Kidney, testis, muscle and spleen showed a decreasing trend (P < 0.05) in AA content with decreasing Se in the diet. Dehydroascorbic acid concentrations were decreased (P < 0.05) in several tissues with reduction in dietary Se (heart and spleen) or AA (liver, heart, kidney, muscle and spleen). At week 12, combined dietary restriction of Se and AA decreased AT concentrations in most tissues. In addition, restriction of Se (liver, heart and spleen) and AA (liver, kidney and spleen) separately also reduced AT in tissues., Conclusion: Together, these data demonstrate sparing effects of Se and AA on vitamin C and AT in guinea pig tissues.
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- 2007
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38. Manganese content of soy or rice beverages is high in comparison to infant formulas.
- Author
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Cockell KA, Bonacci G, and Belonje B
- Subjects
- Consumer Product Safety, Female, Humans, Infant, Male, Manganese adverse effects, No-Observed-Adverse-Effect Level, Nutritional Requirements, Spectrophotometry, Atomic, Beverages analysis, Infant Formula chemistry, Infant Nutritional Physiological Phenomena, Manganese administration & dosage, Oryza chemistry, Soy Milk chemistry
- Abstract
Objective: Well-meaning but inadequately informed parents may perceive plant-based beverages such as soy beverages (SB) or rice beverages (RB) as an alternative to infant formula. Manganese (Mn) is an essential mineral nutrient found at high levels in plants such as soy and rice. Excessive Mn exposure increases the risk of adverse neurological effects., Methods: We analysed, by atomic absorption spectrometry, the Mn content of 36 SB, 5 RB, 6 evaporated milks (EM), 14 soy-based infant formulas (SF) and 16 milk-based infant formulas (MF), obtained from commercial outlets in Ottawa, Canada., Results: SB had the highest levels of Mn (16.5 +/- 8.6 micro g/g dry wt, mean +/- s.d.), followed by RB (9.9 +/- 1.7 micro g/g dry wt). Mn levels of individual SB/RB ranged from 2 to 17 times the mean Mn content of SF (2.4 +/- 0.7 micro g/g dry wt) and 7 to 56 times that of MF (0.70 +/- 0.35 micro g/g dry wt). EM contained very little Mn (0.02 +/- 0.03 micro g/g dry wt). Calculated mean Mn intakes from SB/RB by infants up to 6 months of age, assuming complete substitution of these products (0.78 L/day), approached the Tolerable Upper Intake Level (UL) for 1-3 year olds (no UL for Mn is available for infants under 1 year of age). Expressed as micro g Mn/100 kcal, SB/RB exceeded the range derived from ULs and typical energy intakes of 1-3 year olds., Conclusions: SB/RB should not be fed to infants because they are nutritionally inadequate and contain Mn at levels which may present an increased risk of adverse neurological effects if used as a sole source of nutrition.
- Published
- 2004
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39. Interaction of human tissue plasminogen activator (t-PA) with pregnancy zone protein: a comparative study with t-PA-alpha2-macroglobulin interaction.
- Author
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Sánchez MC, Chiabrando GA, Guglielmone HA, Bonacci GR, Rabinovich GA, and Vides MA
- Subjects
- Electrophoresis, Polyacrylamide Gel, Female, Humans, In Vitro Techniques, Pregnancy, Pregnancy Proteins isolation & purification, Protein Binding, Protein Conformation, Sulfhydryl Compounds chemistry, Tissue Plasminogen Activator isolation & purification, src Homology Domains, Pregnancy Proteins metabolism, Tissue Plasminogen Activator metabolism, alpha-Macroglobulins metabolism
- Abstract
Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.
- Published
- 1998
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40. Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases.
- Author
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Ramos A, Remedi MS, Sánchez C, Bonacci G, Vides MA, and Chiabrando G
- Subjects
- Animals, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Endopeptidases drug effects, Endopeptidases isolation & purification, Leupeptins pharmacology, Protease Inhibitors metabolism, Time Factors, Tosyl Compounds pharmacology, Trypanosoma cruzi drug effects, alpha-Macroglobulins metabolism, Endopeptidases metabolism, Protease Inhibitors pharmacology, Trypanosoma cruzi enzymology, alpha-Macroglobulins pharmacology
- Abstract
The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.
- Published
- 1997
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41. A procedure for human pregnancy zone protein (and human alpha 2-macroglobulin) purification using hydrophobic interaction chromatography on phenyl-sepharose CL-4B column.
- Author
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Chiabrando G, Bonacci G, Sanchez C, Ramos A, Zalazar F, and Vides MA
- Subjects
- Chelating Agents, Electrophoresis, Polyacrylamide Gel, Female, Humans, Pregnancy, Pregnancy Proteins chemistry, Protein Conformation, Sepharose analogs & derivatives, Zinc, alpha-Macroglobulins chemistry, Chromatography, Agarose methods, Pregnancy Proteins isolation & purification, alpha-Macroglobulins isolation & purification
- Abstract
In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl-Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human alpha 2-macroglobulin (alpha 2-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of alpha 2-M from the same pregnancy plasma, based on the differential elution of PZP and alpha 2-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP-protease complexes, HIC on phenyl-Sepharose column could also be used for separating both conformational states of PZP.
- Published
- 1997
- Full Text
- View/download PDF
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