Changes In Nitric Oxide Levels Research Articles

Real-time and specific monitoring of nitric oxide and evaluating of the oxidative stress in living cells and zebrafish under the pollutant exposure using a carbon dot-based composite fluorescent probe

Nitric oxide (NO) functions as an essential signalling molecule in various physiological and pathological pathways. In vitro and vivo redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) directly influence the intracellular state. In this study, a red-emitting fluorescent nanoprobe, N,S-CDs@Zn-ICA, was synthesized to monitor NO fluctuations in living cells and zebrafish under the exposure to various pollutants. Red-emissive carbon dots (N,S-CDs) were synthesized by a hydrothermal method using o-phenylenediamine and urea as carbon / nitrogen sources, and H2SO4 as sulfur source. Glutathione (GSH) was introduced to link N,S-CDs with metal organic complexes (Zn-ICA) through an amidation reaction to fabricate a carbon dot-based composite fluorescent probe, which greatly improved the selectivity, stability, and response time of the N,S-CDs. The composite probe has high selectivity and sensitivity with limit of detection (LOD) of 96.0 nM. Furthermore, the proposed probe was successfully used to monitor the dynamic changes in NO levels and evaluate oxidative stress in MCF-7 cells and zebrafish under the exposure to various pollutants, including seven heavy metal ions (such as Pb2+, Cd2+, and Hg2+) and nine organic pollutants at different concentrations and exposure times. This work provides a novel strategy for constructing highly selective and red-emitting fluorescent probe for real-time and dynamic monitoring of NO and further evaluating oxidative stress induced by pollutants in vitro and in vivo via fluorescence imaging.

A Manganese(Ⅱ) complex as high-order multiphoton fluorescent probe for detecting nitric oxide content change in idiopathic pulmonary fibrosis mice

Nitric oxide (NO) is an important gas signaling molecule, is present in the alveoli in correlation with the extent of idiopathic pulmonary fibrosis (IPF) patients. In this work, a high-order multiphoton fluorescent probe FS-MnCl2 is designed and synthesized for detecting NO level in mice model of IPF. FS-MnCl2 has the advantage of low detection limit (11 nM) and resistance to photobleaching in vitro. The three-photon absorption cross section is greatly enhanced after FS-MnCl2 recognizes NO. Moreover, the fluorescence intensity of FS-MnCl2-NO is stronger than that of FS-MnCl2 in the presence of RNA. Biological applications suggest that FS-MnCl2 can monitor changes in NO levels in living cells and IPF mice. This work provides a new strategy for reasonably designing Mn(Ⅱ) complex that can respond to NO for predicting IPF at an early stage.

Changes in Nitric oxide Levels in Basal Forebrain during Sedation with Dexmedetomidine and Sevoflurane

BackgroundPerioperative and critical care settings frequently require periods of prolonged sedation but the effect of sedation on sleep homeostasis is not completely understood. In the ongoing studies in our laboratory, continuous 8 h sedation with dexmedetomidine (DEX) was shown to increase wakefulness (WAKE), decrease slow‐wave sleep (SWS), and nearly eliminate rapid eye movement sleep (REMS) in the post‐sedation period [1]. In contrast, continuous 8 h sedation with sevoflurane (SEVO) did not affect the time spent in WAKE or SWS, but increased REMS in the post‐sedation period [1]. Past studies have shown nitric oxide (NO) in basal forebrain (BF) to be a key modulator of sleep homeostasis. Specifically, NO concentration in BF has been shown to increase with sleep pressure (e.g., during sleep deprivation) and decrease with the reduced sleep need (e.g., during recovery from sleep deprivation). To determine the effect of sedation produced by DEX or SEVO on sleep homeostasis, as inferred from changes in NO levels in BF, we quantified the changes in NO concentration before, during, and after sedation with DEX and SEVO.MethodsAdult male Sprague Dawley rats (N=4 male) were surgically prepared to record electroencephalogram (EEG) and electromyogram and intravenous delivery of DEX via a chronic catheter in jugular vein. In addition, an open flow microperfusion guide cannula was implanted into BF (N=2) or prefrontal cortex (N=2) for collection of perfusates for NO analysis. Prefrontal cortex (PFC) was used as an anatomical control for BF. After at least 15 days of post‐surgical recovery, rats were connected to the EEG recording system and a sampling insert was lowered into BF or PFC to route the perfusate samples directly into a high‐performance liquid chromatograph for online analysis of nitrite and nitrate levels, which are collectively termed as NOx and are considered a surrogate for endogenous NO concentration. The EEG and NOx were recorded during 2 h baseline wake condition, 8 h of sedation with either DEX (0.5‐0.7ug/kg/min) or SEVO (1.5‐3.0%), and 2 h post‐sedation period. Each rat was administered with DEX or SEVO in a counter‐balanced manner and with a washout period of at least 7 days.ResultsThe NOx levels in BF decreased during sedation with DEX (mean ± standard deviation: 0.52 μM ± 0.31 during baseline vs 0.08 μM ± 0.01 during DEX) and SEVO (mean ± standard deviation: 1.58 μM ± 0.16 during baseline vs 0.72 μM ± 0.07 during SEVO). The NOx levels in PFC during sedation with DEX stayed about at the same level as observed during baseline state (2.40 μM ± 1.39 during baseline vs. 3.01 μM ± 0.16 during DEX), but the NOx levels in PFC decreased during sedation with SEVO (1.45 μM ± 1.23 during baseline vs 0.37 μM ± 0.18 during SEVO).ConclusionAlthough these are preliminary data, the decrease in NOx levels in BF after sedation with DEX and SEVO suggest a reduced sleep pressure and is supportive of the changes in sleep homeostasis as described in our previous report.References Parkar A, Liu T, Fryzel A, Groenhout T, Mashour GA and Pal D (2020) Effect of prolonged sedation with dexmedetomidine, midazolam, propofol, or sevoflurane on sleep homeostasis in rat. J Neurosurgical Anesthesiology, Vol 32, No 4, Page 403, Abstract no SNACC‐387.

Estrous cycle phase affects myocardial infarction through reactive oxygen species and nitric oxide.

Myocardial infarction is the leading cause of death in women worldwide. Several studies have shown that estrogens play a cardioprotective role in women by decreasing reactive oxygen species (ROS) and increasing nitric oxide (NO). The aim of this work was to determine whether the evolution of myocardial infarction depends on the phase of the estrous cycle. Female Wistar rats were randomized into the following groups with an (n = 7 per group): (1) ovariectomized (OVX-sham); (2) OVX-48 h coronary occlusion (CO); (3) OVX-2 w CO; (4) proestrus-sham; (5) proestrus-48 h CO; (6) proestrus-2 w CO; (7) estrus-sham; (8) estrus-48 h CO; and (9) estrus-2 w CO. We measured the percentage of myocardial necrosis, cardiac hypertrophy, hemodynamic parameters, and the production of NO and ROS, after acute and chronic myocardial infarction was induced in proestrus or estrus or ovariectomized female rats. The infarct area was reduced in the proestrus groups, while it was increased in the estrus and OVX groups. The left ventricular systolic pressure (LVSP) and ± dP/dt were reduced, but left ventricular diastolic pressure (LVDP) was increased in the OVX groups. NO was increased in the OVX + CO and estrus + CO groups. Production of ROS was increased in OVX rats after myocardial infarction but remained unchanged in proestrus and estrus. The phase of the estrous cycle in which the myocardial infarction occurs is important. When the coronary occlusion occurs during the proestrus phase, it prevents changes in cardiac function, the development of hypertrophy, oxidative stress and changes in NO levels, and reduces the extent of infarction.

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.

Characterization of a lignin from Crataeva tapia leaves and potential applications in medicinal and cosmetic formulations

Lignins are phenolic macromolecules that have several applications. In this work, we examine some biological activities of a lignin-like macromolecule isolated from the Crataeva tapia leaves, not yet studied to evaluate its potential applications in medicinal and cosmetic formulations. Lignin was obtained by alkaline delignification and its physical-chemical characterization was made by means of FT-IR, UV–Vis, NMR spectroscopy, elementary analysis, molecular mass determination and thermal analysis. Lignin is of the GSH type, with levels of hydrogen (5.10%), oxygen (27.18%), carbon (67.60%), nitrogen (0.12%) and phenolic content of 189.6 ± 9.6 mg GAE/g. In addition, it is a thermally stable macromolecule with low antioxidant activity. Cytotoxicity and cytokine production were assessed by flow cytometry. The photoprotective activity was evaluated by adding different concentrations of lignin to a commercial cream. Lignin was not cytotoxic, it stimulated the production of TNF-α, IL-6 and IL-10 and did not promote a significant change in nitric oxide levels. In addition, this macromolecule was able to promote increased absorption of ultraviolet light from a commercial cream. These results reinforce the ethnopharmacological use of C. tapia leaves and suggest the need for further studies to determine the potential medicinal and cosmetic applications (sunscreen) of lignin from C. tapia leaves.

Oral L-Arginine lower neovascularization and the number of fibroblasts but failed to increase nitric oxide and epithelialization in the healing process of male white diabetic wistar rats (Rattus norvegicus)

Introduction: Oral L-Arginine is a conditional essential amino acid that plays a role in wound healing in DM. The role of arginine in diabetic wounds is by enhancing blood circulation in the injured area and increasing oxygen supply to the wound tissue. The purpose of this study to prove the administration of oral L-Arginine toward vascularization status in wound healing of male white rats wistar diabetes mellitus.
 Methods: A randomized posttest only control group study using with 36 diabetic induced wistar rats (Rattus Norvegicus) aged 2-3 months and weighing 180-200gram which then divided randomly into two groups. Nitric oxide level was measured on the third day and each group was then further divided into two groups for examination of neovascularization, fibroblasts and epithelialization on the seventh day and on the tenth day.
 Results: Administration oral L-Arginine failed to induce any significant change in Nitric Oxide level and wound gap closure. On the other hand, the results showed that the mean neovascularization was significantly different between the two groups on the 10th day (Control group vs intervention group: 4.22±1922 vs1.89±1364; p=0.009). In addition, the mean number of fibroblast at the 10th day was also significantly different (Control group vs intervention group: 74.11±28.57 vs 38.11±20.90; p=0.008).
 Conclusion: In conclusion, oral L-Arginine did not significantly affect nitric oxide and epithelialization while decreased neovascularization and the number of fibroblasts on day tenth in the healing process of male white rats diabetes mellitus

Hydrogen peroxide-based products alter inflammatory and tissue damage-related proteins in the gingival crevicular fluid of healthy volunteers: a randomized trial

Hydrogen peroxide (H2O2)-based products are effective in tooth whitening; however, their safety is controversial as they may harm patient tissues/cells. These effects are suggested to be concentration-dependent; nonetheless, to date, there are no reports on H2O2-mediated oxidative damage in the gingival tissue, and neither whether this can be detected in gingival crevicular fluid (GCF) samples. We hypothesize that H2O2 whitening products may cause collateral oxidative tissue damage following in office application. Therefore, H2O2 and nitric oxide (NO) levels were investigated in GCF samples obtained from patients undergoing dental bleaching with H2O2 at different concentrations, in a randomized, double-blind, split-mouth clinical trial. A proteomic analysis of these samples was also performed. H2O2-based whitening products promoted inflammation which was detected in GCF samples and lasted for longer following 35% H2O2 bleaching. This included time-dependent changes in NO levels and in the abundance of proteins associated with NO synthesis, oxidative stress, neutrophil regulation, nucleic acid damage, cell survival and/or tissue regeneration. Overall, H2O2-based products used in office promote inflammation irrespective of their concentration. As the inflammation caused by 35% H2O2 is longer, patients may benefit better from using lower concentrations of this bleaching product, as they may result in less tissue damage.

Exogenous ascorbic acid is a pro-nitrant in Arabidopsis thaliana

Due to the intensified production of reactive nitrogen species (RNS) proteins can be modified by tyrosine nitration (PTN). Examination of PTN is a hot topic of plant biology, especially because the exact outcome of this modification is still pending. Both RNS and ascorbic acid (AsA) are redox-active molecules, which directly affect the redox state of cells. The possible link between RNS-dependent PTN and AsA metabolism was studied in RNS (gsnor1-3, nia1nia2) and AsA (vtc2-3) homeostasis Arabidopsis mutants. During physiological conditions, intensified PTN was detected in all mutant lines compared to the wild-type (WT); without altering nitration pattern. Moreover, the increased PTN seemed to be associated with endogenous peroxynitrite (ONOO-) levels, but it showed no tight correlation with endogenous levels of nitric-oxide (NO) or AsA. Exogenous AsA caused intensified PTN in WT, vtc2-3 and nia1nia2. In the background of increased PTN, significant NO and ONOO- accumulation was detected, indicating exogenous AsA-induced RNS burst. Interestingly, in AsA-triggered stress-situation, changes of NO levels seem to be primarily connected to the development of PTN. Our results point out for the first time that similarly to human and animal systems exogenous AsA exerts pro-nitrant effect on plant proteome.

Effect of alpha-lipoic acid supplementation on blood pressure, renal oxidant-antioxidant status and renal damage in spontaneously hypertensive rats

Objective: To investigate the effect of alpha-lipoic acid (ALA) supplementation on systolic blood pressure (SBP), renal oxidant-antioxidant status and renal damage in spontaneously hypertensive rats (SHR) and SHR administered with Nω-nitro-L-arginine methyl ester (L-NAME). Methods: Male rats were divided into four groups (SHR, SHR+ALA, SHR+L-NAME, SHR+ALA+L-NAME). The respective group of rats was administered with ALA (100 mg/ kg/day) from age 4 weeks to 28 weeks and L-NAME (25 mg/kg/day) from age 16 weeks to 28 weeks. SBP was measured every two weeks and twenty four hour urine was collected at 4 weeks, 16 weeks and 28 weeks for estimation of protein, creatinine and N-acetyl-β-D-glucosaminidase. At the end of 28 weeks, rats were sacrificed and blood and kidneys collected for assessment of blood creatinine, kidney thiobarbituric acid reactive substances, protein carbonyls, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glutathione disulfide, glutathione, total antioxidant status and nitric oxide as well as histopathological examination. Results: ALA supplementation significantly reduced SBP of SHR and SHR+L-NAME rats when compared to their respective non-supplemented groups. Renal oxidant status markers including thiobarbituric acid reactive substances and protein carbonyls were significantly reduced on SHR and SHR+L-NAME rats supplemented with ALA at 28 weeks as well as ALA supplementation significantly increased renal antioxidants including superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione and glutathione/ glutathione disulfide ratio at 28 weeks. No significant change in nitric oxide levels was observed between the ALA supplemented and non-supplemented groups. Renal dysfunction was ameliorated on ALA supplementation as evidenced by significant reduction in urine protein levels, N-acetyl-β-D-glucosaminidase activity and significant increase of creatinine clearance in SHR and SHR+L-NAME at 28 weeks. Renal histopathological examination showed that ALA supplementation prevented vascular damage in SHR and ameliorated glomerular damage in SHR+L-NAME at 28 weeks. Conclusions: ALA has hypotensive and renoprotective effects on both SHR and SHR+L-NAME, which could be due to its ability to ameliorate oxidative stress in the kidneys.

Microelectrophoretic single-cell measurements with microfluidic devices.

Here we describe in detail the design, fabrication and operation of our automated high-throughput single cell microchip electrophoresis device with laser induced fluorescence detection. Our device features on-board integrated peristaltic pumps that generate flow directly within the microfluidic channels. Additionally, we have incorporated an optical fiber bridge that enables simultaneous fluorescence detection at two points of interest within the device without the need for additional optical components or detectors. The second detection spot is used to detect the intact cell immediately prior to lysis giving a signal at t=0s for each single-cell electropherogram. We can also use this signal to measure the absolute migration time of the separated analytes to confidently determine the identity of each peak. Finally, we demonstrate the application of our device for the measurement of intracellular nitric oxide (NO) levels in T-lymphocytes. Changes in NO levels within cells is associated with a number of chronic diseases including neurodegenerative, cardiovascular and cancers. We show that our system is capable of measuring NO levels under the following conditions: native, lipopolysaccharide stimulation, and inhibition of inducible nitric oxide synthase. It is our hope that the information and procedures described in this chapter may enable others to use or adapt our system for other analyses at the single cell level.

A17266 Intradialytic Hypertension and Its Association with Nitric Oxide Level Changes

Objectives: Intradialytic hypertension is associated with increased morbidity and mortality, yet the mechanism is uncertain. Prior studies investigate imbalances in endothelial-derived vasoregulators nitric oxide, which these studies show conflicting result. The aim of the study was to investigate the association between intradialytic hypertension and the changes of nitric oxide level pre-to-post-dialysis. Methods: We conducted a before-and-after experimental studies in hemodialysis unit of Mohammad Hoesin General Hospital Palembang including 12 hemodialysis subjects without (controls) and 12 with intradialytic hypertension (an increased systolic BP pre-to-post-dialysis ≥10 mmHg in ≥4/6 consecutive HD sessions). The primary outcome was the changes of nitric oxide pre-to-post-dialysis, which was assessed in the 7th HD session, using ELISA. Dependent t-test was used to compare nitric oxide level changes. Results: Baseline characteristics, comorbidities, and anti-hypertensive drug consumption were similar between groups. Compared with controls, nitric oxide level changes was significantly higher among subjects with intradialytic hypertension (14.21 ± 10.24 u/M vs 6.45 ± 5.00 u/M). The changes of nitric oxide level pre-to-post-dialysis between 2 groups showed mean difference of 9.14 ± 2.06 u/M and 95%CI value was 4.87–13.41. Conclusion: Intradialytic hypertension is associated with nitric oxide level changes. We propose that nitric oxide level changes pre-to-post-dialysis may partially explain the higher event rates observed in these patients.

Excessive Ultrafiltration During Hemodialysis Plays a Role in Intradialytic Hypertension Through Decreased Serum Nitric Oxide (NO) Level

Background: Intradialytic hypertension is one of many complications during Hemodialysis (HD). The mechanism of intradialytic hypertension is currently unclear. Objective: This research aims to understand the association between excessive Ultrafiltration (UF) and intradialytic hypertension episode and its relationship with changes in endothelin-1 level (ET-1), Asymmetric Dimethylarginine (ADMA) level and Nitric Oxide (NO) level during HD. Methods: This study utilized a case-control design. A sample of one hundred and eleven patients who were already undergoing maintenance HD for more than three months was included. Serum levels of NO, ET-1, and ADMA were examined before and after HD; samples were followed by as much as six times consecutive HD session, in which ultrafiltration and blood pressure during HD were noted. Results: From 112 samples obtained, 32.1% (36/112) had intradialytic hypertension. Using regression analysis, we found a significant association between changes in NO levels and intradialytic hypertension. We found a significant association between excessive UF and intradialytic hypertension (p=0.001), adjusted OR=5.17. Path analysis showed the existence of a significant relationship between UF volume during HD and intradialytic hypertension (CR 5.74; p<0.01), as well as a significant relationship between UF volume during HD and NO levels (CR -3.70: p<0.01). There was a direct relationship between NO serum levels with intradialytic hypertension (CR -7.08: p<0.01). Conclusion: Excessive UF during HD plays a role in intradialytic hypertension episode through decreased NO serum levels. There was no clear role of ADMA and ET-1 serum levels on intradialytic hypertension episode.

Decreased serum level of nitric oxide in children with excessive body weight

Nitric oxide (NO) is an important mediator involved in vascular homeostasis. Changes in NO level are considered to be associated with obesity and its clinical consequences. Previous studies on NO levels in obese children provided inconclusive results, so this issue requires clarification. One of the main goals of this study was to assess whether childhood excessive body weight (EBW) is associated with changes in serum NO level and whether features like age and gender are linked to NO levels in selected groups. In the present study, the serum NO levels were compared in 43 children with EBW and 37 ageand gender-matched children with normal weight. Moreover, in both groups, body measurements and various clinical parameters, including the serum concentrations of arginine (Arg), a precursor of NO, were determined. The mean serum NO level in EBW group (8.7 ±3.1 μmol/L) was significantly lower than in normal weight group (22.2 ±11.5 μmol/L). However, the serum Arg concentrations were higher in EBW children than in controls. Serum asymmetric dimethylarginine (ADMA) levels were higher in EBW group and inversely correlated with serum NO. The EBW female subgroup was characterized by slightly lower level of NO than the EBW male subgroup. There were no significant changes in serum NO level among different age subgroups in both groups. Our results revealed that EBW in children is associated with significantly decreased level of serum NO. The decreased serum NO level in EBW children is not a result of depleted Arg in the blood. Asymmetric dimethylarginine may at least partially contribute to decreased NO levels in children with EBW. A decreased level of NO could be a potential early marker of the risk of cardiovascular disorders developing in children with EBW.

Structural and lipid peroxidation effects of lead on rat hippocampus and its attenuation by hydrogen rich water

Despite the well-known toxicity and the efforts to control its exposure, lead still has a serious health concern, particularly in young ages. Chelation therapy cannot correct the neurocognitive effects of chronic exposure. So, there is a requirement to test different protective agents for lead intoxication. Hydrogen-rich water (HRW) has gained attraction recently as an antioxidant. Four groups of rats received sodium acetate, HRW, lead acetate (LA), or LA plus HRW for 8 weeks. Oxidative stress, histological and immunohistochemistry using p53 antibody were used to investigate the toxic effect of lead and the possible HRW protective effect in rat hippocampus. Results showed that HRW corrected the elevated malondialdehyde levels (MDA) and restore the lead-induced depletion of antioxidant enzymes; glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD). HRW does not affect the diminished nitric oxide (NO) level in the LA-treated group. Moreover, HRW reversed the LA-induced histological and immunohistochemical changes. It significantly decreased the percentage of the apoptotic index. We concluded that HRW protects the neurons against lead-induced oxidative stress and has anti-apoptotic effects without a noticeable change in NO level which already was diminished by LA.

Measurement of nitric oxide bioavailability during dynamic handgrip exercise in women with Metabolic Syndrome.

BackgroundThe bioavailability of nitric oxide (NO) has been shown to contribute to vascular function during peak exercise. Young women with Metabolic Syndrome (MetSyn) have presented with a deficit in vascular conductance during exercise hyperemia. Since NO has been shown to play a key role in vasodilation during exercise, it is crucial to identify if there is a deficit in the NO synthesis and how it affects vasculature of these young women. The aim of this study is to measure changes in bioavailable blood NO levels at rest and during peak handgrip exercise and determine its correlation to vascular conductance in women with MetSyn compared to healthy controls.MethodsIn this study, 15 participants (6 MetSyn and 9 Controls) performed the procedure of graded handgrip exercise on a dynamic handgrip device while beat‐to‐beat blood pressure (CNAP finger plethysmography), brachial artery diameter and blood flow with Doppler ultrasound and B‐mode imaging were measured continuously. During this exercise, participants used a handgrip dynamometer at a cadence of 30 contractions/minute. Exercise workload was increased in a ramp fashion (0.5 kg·min−1) until task failure. At rest and immediately upon task failure, a venipuncture was performed to take rest and peak NO levels to be later measured by a EPR Spectroscopy and Colorimetric Nitrate/Nitrite commercial ELISA kit. Whole blood was drawn into a prepared vacutainer in a 1:1 venous blood to the metal chelator, deferoxamine (DF), in diethyldithiocarbamate (DETC) Krebs buffer. Plasma was preserved with EDTA and all samples were immediately flash frozen in liquid nitrogen and stored at −80 degrees Celsius.ResultsThere was a significant difference in resting NO values (p=0.034) as well as end NO concentration values (p=0.053) between MetSyn and control groups. However, the MetSyn showed higher resting and peak NO values than control group as measured by ELISA. The change in plasma NO did not show any significant changes from rest to peak exercise in both groups (MetSyn p=0.757, control p=0.562). However, using the EPR spectroscopy method there is a significant difference identified between rest and peak exercise (p<0.05). There was no significant change in NO between the groups (p=0.633). The MetSyn group has a significantly diminished arterial conductance in the brachial artery during handgrip as well as a attenuated response in the femoral artery during dynamic leg kick. The change in brachial conductance did not show significant correlation to the change in NO levels in either of the groups (MetSyn p=0.792, r=−0.321, control p=0.549, r=−0.310) with the ELISA method.ConclusionsThe colorimetric nitrate/nitrite ELISA assay is not a reliable technique to measure the degree of change of NO concentrations at peak exercise. The whole blood EPR technique previously used in our laboratory work showed more significant results associated with the declining exercise hyperemia of the MetSyn group. Future studies will focus on clarifying methodologies for quantifying blood NO bioavailability at peak exercise.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in nitric oxide levels and their relationship with callose deposition during the interaction between soybean and Soybean mosaic virus.

The present study aimed to investigate changes in nitric oxide (NO) level and its relationship with callose deposition during the interaction between soybean and Soybean mosaic virus (SMV). Soybean cv. 'Jidou 7' and SMV strains N3 and SC-8 were used to constitute incompatible and compatible combinations. Intracellular NO was labelled with the NO-specific fluorescence probe DAF-FM DA. Confocal laser scanning microscopy (CLSM) was then used to observe changes in NO production during SMV infection-induced defence responses in soybean. The results showed NO fluorescence increased rapidly at 2-72h post-inoculation, peaked at 72h and then decreased in the incompatible combination. However, in the compatible combination, extremely weak NO fluorescence appeared in the early stage (2-24h) post-inoculation, but was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 or 72h post-inoculation. The same occurred when injections of NR or NOS inhibitors were applied prior to inoculation. The observation of callose fluorescence in the incompatible combination revealed that either the elimination or reduction of NO in the early stage led to a delay in callose formation, enabling the virus to cause systemic infection. Together with our previous findings, this study indicates that viral infection could induce NO production and callose deposition during the incompatible interaction between soybean and SMV. The production of NO involves NR and NOS enzymatic pathways, and NO mediates the process of callose deposition at plasmodesmata.

Deletion of T-type calcium channels Cav3.1 or Cav3.2 attenuates endothelial dysfunction in aging mice.

Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Cav3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Cav3.1, and Cav3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Cav3.2-/- preparations but increased in those of Cav3.1-/- mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Cav3.1-/- and Cav3.2-/- preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Cav3.1-/- and Cav3.2-/- mice. The expression of Cav3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Cav3.1-/- mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Cav3.1-/- mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.

Protective Effect of Dark Chocolate on Cardiovascular Disease Factors and Body Composition in Type 2 Diabetes: A Parallel, Randomized, Clinical Trial

Background: Diabetes leads to complications such as cardiovascular diseases. There are limited data about the effect of dark chocolate on cardiovascular function in patients with diabetes. Objectives: The current study aimed at determining the effect of dark chocolate on cardiovascular health and body composition among people with diabetes. Methods: The current parallel, randomized, clinical trial was conducted on 44 patients with diabetes (Ahvaz, Iran). They were randomly assigned into the intervention (n = 21, 30 g dark chocolate daily for 8 weeks) and the control groups (n = 23). At the beginning and end of the intervention period, fasting blood samples were collected to measure nitric oxide (NO) and angiotensin II. Also, anthropometric measurement, body composition analyses, and blood pressure were compared between the 2 groups before and after the intervention. Results: A significant reduction in systolic (-6.9 ± 7.3 vs. 0.3 ± 1.9; P = 0.001) and diastolic blood pressure (-5.8 ± 6.7 vs. 0.5 ± 3.9; P = 0.001), waist circumference (WC) (-0.7 ± 1.0 vs. 0.1 ± 1.2; P = 0.007), and significant increase in soft lean mass (P = 0.045) was observed in the intervention group. There were no significant changes in NO levels, but a trend close to significance for angiotensin II (P = 0.052) at end of the intervention between the 2 groups. Conclusions: The current study findings showed that dark chocolate consumption in patients with diabetes might improve their WC, body composition, and blood pressure, but had no effect on NO in this dosage.

Serum Nitric Oxide as a Predictive Biomarker for Bevacizumab in Non-small Cell Lung Cancer Patients.

Reportedly, hypertension tends to be associated with response to bevacizumab therapy, because bevacizumab suppresses vascular nitric oxide production. In this study we examined the predictive value of nitric oxide in bevacizumab-treated non-small cell lung cancer (NSCLC) patients. Fifteen patients with advanced or recurrent NSCLC treated with bevacizumab-based regimens were evaluated retrospectively. Serum NOx (NO2-/NO3-) was assayed by the Griess method. Serum nitric oxide levels were decreased after two courses of bevacizumab treatment in our responder group (p=0.02). According to the change in nitric oxide levels after the second course of treatment, median progression-free survival was 11.0 months in the group with decreased serum nitric oxide and 7.6 months in the group with increased serum nitric oxide (p=0.08). Serum nitric oxide levels could be a predictive biomarker for response to bevacizumab in NSCLC patients.