Journal: Food Bioscience

Article Title: Anti-inflammatory and Antioxidative Effects of Bioactive Peptides IKW and RIY in Spontaneously Hypertensive Rats and Angiotensin II-Stimulated Vascular Smooth Muscle Cells

doi: 10.1016/j.fbio.2024.105417

Figure Lengend Snippet: Fig. 4. Peptides alleviated excessive oxidative stress in the aorta. Panel A–B: Immunostaining of nitrotyrosine in aortic sections. Panel C–D: Immunostaining of intracellular Ca2+ levels in aortic sections using Fluo-4 AM (n = 4–6). ** and **** indicate p < 0.01 and p < 0.0001, respectively, compared to the untreated control (Untr).

Article Snippet: They were then incubated overnight at 4 ◦C with a 1:20 dilution of nitrotyrosine primary antibody (Novus biologicals, Oakville, ON, Canada).

Techniques: Immunostaining, Control

Journal: Frontiers in Neurology

Article Title: Oxidative imbalance as a co-player in jaw functional limitations and biopsychosocial profile in patients with temporomandibular disorder—myofascial pain with referral

doi: 10.3389/fneur.2024.1509845

Figure Lengend Snippet: Multiple linear regression model with the BDI as the dependent variable and saliva concentrations of kynurenine, TOS, nitrotyrosine, GSH, and UA as independent variables.

Article Snippet: The commercial kit Nitrotyrosine ELISA Immundiagnostik AG (Bensheim, Germany) supported by the manufacturer’s instructions was used.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Selective peroxynitrite-mediated protein nitration catalyzed by glyoxalase domain containing protein 4

doi: 10.1073/pnas.2515002123

Figure Lengend Snippet: Screening for tyrosine nitration activity. ( A ) Candidate proteins were expressed using the in vitro transcription/translation (IVTT) platform. The synuclein nitration assay was conducted by adding 1 µg of α-syn peptide (biotinylated α-syn-Y133,136), 4 µg of IVTT-expressed protein lysate, and 100 µM peroxynitrite for 10 min. ELISA with streptavidin capture and detection with nSYN12, a specific monoclonal antibody that recognizes nitrated Y125 and Y136-α-syn, quantified the nitration levels of the α-syn peptide (OD450). Candidate proteins that showed a level of α-syn nitration above that of the IVTT control lysate were considered potential candidates for α-syn. The values represent the mean ± SEM of n = 2 for this representative experiment. Similar results were obtained from 3 different experiments using IVTT-expressed protein lysates as a source of synuclein nitration activity. ( B ) Recombinantly expressed and purified GLO2 (20 ng) and GLOD4 (10 pg) were tested for their ability to nitrate the α-syn peptide (biotinylated a-syn-Y133,136). The values are expressed as the fold change from the level of nitration by 100 µM peroxynitrite in the absence of GLO2 or GLOD4 and represent the mean ± SEM of n = 2 in this representative experiment. Similar results were obtained from 3 different experiments. ( C ) Substrate specificity for GLOD4-catalyzed protein tyrosine nitration. The nitration of potential substrates was assessed via a HuProt microarray chip (CDI) containing 23,000 unique human proteins in the presence of 200 ng of GLOD4 and 2 consecutive boluses of 100 µM peroxynitrite. The levels of nitrated proteins were quantified by ELISA using a cocktail of anti-nitrotyrosine antibodies ( Materials and Methods ). The figure and table depict values obtained in duplicate per chip. Three separate experiments were conducted using 3 different chips with the same results obtained. GLOD4 catalyzed the nitration of α-syn and β-syn, PPM1B and PQBP1. The statistical analysis parameters for this test are listed below in the graph for the top 4 putative substrates. F635 is the average foreground signal intensity of 2 replicate spots for a given protein in the detection channel (635 nM). Z = [(F635-F635(ave)]/F635(SD); F635(ave) and F635(SD) are the average and SD of the F635 values of all spots on the chip; the S score is the difference in the Z scores for a given protein and those ranked in its surrounding area. If the S score of the hit was > 3, the score was considered highly specific. n > 3 experiments. The four proteins with Z scores > 10 and S scores > 3 were considered putative substrates. ( D ) Independent confirmation of the GLOD4 nitration substrates using 1 µg of substrate, 4 ng of GLOD4, and 100 µM peroxynitrite in a 40 µL reaction for 10 min. The substrates were captured via an anti-His antibody, and tyrosine nitration was quantified via ELISA using a cocktail of anti-3-nitrotyrosine antibodies. The values represent the mean ± SD of n = 4 and are expressed as the fold change relative to the nitration level without GLOD4.

Article Snippet: GLOD4 nitration was detected via western blotting using a mouse monoclonal antibody against nitrotyrosine (clone 39B6, Novus Biologicals #NB110-96877).

Techniques: Nitration, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Control, Purification, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Selective peroxynitrite-mediated protein nitration catalyzed by glyoxalase domain containing protein 4

doi: 10.1073/pnas.2515002123

Figure Lengend Snippet: Autonitration of other putative protein catalysts of nitration. ( A ) The autonitration of GLOD4, GLO1, GLO2, GLOD5, HPD, MBLAC2, and HAGHL was determined by western blotting. Lane 1: unreached protein, Lane 2: protein (1 µg) reacted with 100 µM peroxynitrite (50 µM for HAGHL), and Lane 3: protein reacted with 100 µM peroxynitrite in the presence of 30 µM NB001 (100 µM NB001 for HAGHL). The proteins were detected by an anti-His (clone HIS.H8), anti-Flag (clone M2), or anti-HAGHL (clone 3H2) antibody ( Top row), and nitration was detected using an anti-3-nitrotyrosine antibody ( Bottom row). ( B ) MBLAC2-mediated nitration of PIGR, and MCEE-mediated nitration of creatine kinase (CKMT1A). Protein tyrosine nitration activity was determined by incubating each enzyme (1, 5, or 10 ng) with 2 µg of substrate and 100 µM peroxynitrite with or without 100 µM NB001 for 10 min. Experiments were repeated three times with similar results.

Article Snippet: GLOD4 nitration was detected via western blotting using a mouse monoclonal antibody against nitrotyrosine (clone 39B6, Novus Biologicals #NB110-96877).

Techniques: Nitration, Western Blot, Activity Assay

Journal: Journal of the International Society of Sports Nutrition

Article Title: Effects of powdered Montmorency tart cherry supplementation on an acute bout of intense lower body strength exercise in resistance trained males

doi: 10.1186/s12970-015-0102-y

Figure Lengend Snippet: Markers of Free Radical Production and Oxidative Stress

Article Snippet: Serum samples were assayed using standard commercially available ELISA kits for Superoxide Dismutase (SOD Activity Assay kit), Total Antioxidant Status (TAS, Antioxidant Assay kit), Thiobarbituric Acid Reactive Substance (TBARS, Malondialdehyde-MDA, TCA method kit) ( Cayman Chemical Company, Ann Arbor, MI, USA ), and Nitrotyrosine ( ALPCO Diagnostics, Salem, NH, USA ).

Techniques:

Journal: Frontiers in physiology

Article Title: Sex-Specific Effects of Nanoparticle-Encapsulated MitoQ (nMitoQ) Delivery to the Placenta in a Rat Model of Fetal Hypoxia.

doi: 10.3389/fphys.2019.00562

Figure Lengend Snippet: FIGURE 1 | Effects of nMitoQ treatment on placental superoxide, peroxynitrite, and NO levels in normoxic and hypoxic placentae of both male and female offspring. Normoxic and hypoxic dams were treated with nMitoQ or saline, and superoxide levels were assessed by DHE staining in placentae from male (A) and female (D) fetuses; peroxynitrite levels were detected by staining for nitrotyrosine (the footprint of peroxynitrite production) in placentae from male (B) and female (E) fetuses, and NO levels were assessed by DAF-FM staining in placentae from male (C) and female (F) fetuses on GD21. Data are represented as mean ± SEM. a.u.: arbitrary units. All groups were compared using a two-way ANOVA followed by Sidak’s post hoc test (n = 6–7/group). *p < 0.05, **p < 0.01 group effect of prenatal hypoxia or nMitoQ treatment; ##p < 0.01, ###p < 0.001 compared to normoxia-saline; ††p < 0.01, †††p < 0.001 compared to hypoxia-saline group.

Article Snippet: Sections were incubated overnight at 4°C with a primary antibody for nitrotyrosine (1:10; mouse-anti-tyrosine, NOVUS Biologicals, Oakville, ON, Canada) or CD31 (1:200 mouse-anti-CD31/PECAM-1, Frontiers in Physiology | www.frontiersin.org 4 May 2019 | Volume 10 | Article 562 NOVUS Biologicals) in 2% BSA/PBS.

Techniques: Saline, Staining