Quantification Of Nitric Oxide Research Articles

Quantification of Nitric Oxide in Single Cells Using the Single-Probe Mass Spectrometry Technique.

Nitric oxide (NO) is a small molecule that plays important roles in biological systems and human diseases. The abundance of intracellular NO is tightly related to numerous biological processes. Due to cell heterogeneity, the intracellular NO amounts significantly vary from cell to cell, and therefore, any meaningful studies need to be conducted at the single-cell level. However, measuring NO in single cells is very challenging, primarily due to the extremely small size of single cells and reactive nature of NO. In the current studies, the quantitative reaction between NO and amlodipine, a compound containing the Hantzsch ester group, was performed in live cells. The product dehydro amlodipine was then detected by the Single-probe single-cell mass spectrometry technique to quantify NO in single cells. The experimental results indicated heterogeneous distributions of intracellular NO amounts in single cells with the existence of subpopulations.

Seasonality impact on the anti-inflammatory, antiproliferative potential and the lignan composition of Bursera microphylla

Bursera microphylla A. Gray (Burseraceae) is a native plant of the Sonoran Desert, used in traditional medicine against different illnesses. The biological potential of B. microphylla has been associated with some groups of bioactive metabolites; amongst the most interesting for its biological effect are the lignan-type compounds. To date, the biological potential of fruits, leaves and stems of B. microphylla has not been widely explored; additionally, it is unknown if the seasons of the year modulate their biological activities and the profile of bioactive compounds. The objective of the present research was to determine the seasonality effect on the anti-inflammatory, antiproliferative potential and the lignan composition of B. microphylla. Ethanolic extracts of stems, leaves and fruits collected during spring, summer, autumn and winter (2019–2020) were obtained. The antiproliferative activity was evaluated against the cell lines C-33 A, HeLa, A549 and ARPE-19 by MTT assay. The anti-inflammatory effect was determined in RAW 264.7-activated with LPS through the quantification of nitric oxide (NO) using the Griess method. The profile of bioactive compounds was investigated by ESI-IT-MSn analysis. Fruits extracts exhibited the highest potential to inhibit the NO production, followed by the stems and leaves extracts. In the same way, fruits extracts displayed the highest antiproliferative activity followed by the stems and leaves extracts. C-33 A was the most sensitive to the treatments, followed by HeLa, A549 and ARPE-19. The chemical analysis allowed the identification of 5 new lignan-type compounds in the extract (hinokinin, 3,4-dimethoxy-3′,4′-methylenedioxylignano-9,9′-epoxylignan- 9′-ol, 7′,8′-dehydropodophyllotoxin, β-peltatin A-methyl ether and 7′, 8′-dehydroacetylpodophyllotoxin). The results show that seasonality modulates the anti-inflammatory, the antiproliferative potential, and the chemical composition of the extracts. The above suggests that B. microphylla could represent a potential source of phytochemicals for the development of pharmacological therapies for inflammation and cancer.

Altered Blood Brain Barrier Permeability and Oxidative Stress in Cntnap2 Knockout Rat Model.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by three core symptoms, specifically impaired social behavior, stereotypic/repetitive behaviors, and sensory/communication deficits. Although the exact pathophysiology of ASD is still unknown, host genetics, oxidative stress, and compromised blood brain barrier (BBB) have been implicated in predisposition to ASD. With regards to genetics, mutations in the genes such as CNTNAP2 have been associated with increased susceptibility of developing ASD. Although some studies observed conflicting results suggesting no association of CNTNAP2 with ASD, other investigations correlated this gene with autism. In addition, CNTNAP2 mediated signaling is generally considered to play a role in neurological disorders due to its critical role in neurodevelopment, neurotransmission, and synaptic plasticity. In this investigation, we studied BBB integrity and oxidative stress in Cntnap2−/− rats. We observed that the BBB permeability was significantly increased in Cntnap2−/− rats compared to littermate wild-type (WT) animals as determined by FITC-dextran and Evans blue assay. High levels of thiobarbituric acid reactive substances and lower amounts of reduced glutathione were observed in brain homogenates of Cntnap2−/− rats, suggesting oxidative stress. Brain sections from Cntnap2−/− rats showed intense inducible nitric oxide synthase immunostaining, which was undetectable in WT animals. Quantification of nitric oxide in brain homogenates revealed significantly high levels in Cntnap2−/− rats compared to the control group. As increased permeability of the BBB and oxidative stress have been observed in ASD individuals, our results suggest that Cntnap2−/− rats have a high construct and face validity and can be explored to develop effective therapeutic modalities.

Microwave-Assisted Synthesis of Schiff Base Metal–Ligand Complexes with Copper and Nickel Centres for Electrochemical In Vitro Sensing of Nitric Oxide in an Aqueous Solution

Nitric oxide (NO), the smallest signalling molecule known in the human body, keeps blood vessels dilated, controls blood pressure, and has numerous other health regulatory effects. The use of Schiff base complexes incorporated onto electrodes to make electrochemical sensors has been explored as an effective method for the determination and quantification of nitric oxide in aqueous solutions. Schiff base ligands were complexed with Cu and Ni metal centres using the microwave synthesis method to produce metal–ligand complexes with enhanced capabilites for NO detection. The electrical current generated at the anode is directly proportional to NO concentrations in the solution through its oxidation to HNO3. Various characterisation techniques were implemented to verify the integrity of each step of metal–ligand synthesis as well as the final product produced, using FT-IR, UV-VIS, and TGA. The as-synthesised Schiff base complexes were electrodeposited on screen-printed carbon electrodes (SPCE) and electrochemically evaluated in a 0.1 M PBS. Furthermore, metal complexes were screened for their in vitro activity towards NO detection in an aqueous solution (PBS). The results show that the investigated sensors (SPCE/Ni-BPND and SPCE/Cu-BPND) respond positively toward NO detection. It was, therefore, identified that the two sensors also do not differ significantly in terms of precision, sensitivity, and lowest detection limit. The sensor strategies demonstrate the NO limits of detection of 0.22 µM and 0.09 µM, and they also demonstrate sensitivity values of 16.3 µA/µM and 13.1 µA/µM for SPCE/Cu-BPND and SPCE/Ni-BPND sensors, respectively.

Metabolic syndrome improves cardiovascular dysfunction and survival during cecal ligation and puncture-induced mild sepsis in mice

AimsSepsis is a potentially fatal systemic inflammatory response and its underlying pathophysiology is still poorly understood. Studies suggest that obesity, a component of metabolic syndrome (MS), is associated with sepsis survival. Therefore, this study focused on investigating the influence of MS on mortality and cardiovascular dysfunction induced by sublethal cecal ligation and puncture (SL-CLP). Main methodsNewborn Swiss mice received monosodium glutamate (MSG) (4 mg kg−1 day−1, s.c.) during the first 5 d of life for MS induction, while the control pups received equimolar saline solution. On the 75th day, SL-CLP was used to induce mild sepsis (M-CLP) in the MS (MS-M-CLP) and control (SAL-M-CLP) mice. The effect of MS on sepsis in mice was assessed by determining the survival rate and quantification of nitric oxide (NO) in the plasma, and associating this data with hematological and cardiovascular parameters. Key findingsMS improved the survival of septic mice, preventing impairment to hematological and cardiovascular parameters. In addition, MS attenuated plasmatic NO increase, which is a typical feature of sepsis. SignificanceThese findings provide new insights into the relationship between obesity and mild sepsis in mice, thus revealing an approach in favor of the “obesity paradox.”

Specific parameters of infrared LED irradiation promote the inhibition of oxidative stress in dental pulp cells

ObjectivesThe present study aimed to assess the oxidative stress and the viability of dental pulp cells stimulated by lipopolysaccharide (LPS) and submitted to photobiomodulation (PBM) with infrared light-emitting diode (LED, 850 nm). DesignThree healthy primary teeth (n = 3) were collected and seeded in 24-well plates with 10 µg/mL of LPS to induce inflammatory mediator formation. The cells were irradiated (850 nm, 40 mW/cm2 and 80 mW/cm2) at the proposed radiant exposures of 0 (control), 4, 15, and 30 J/cm2 shortly after LPS supplementation. The tests were performed 24 h after irradiation to assess mitochondrial activity (MTT assay), the number of viable cells (Trypan Blue), cell morphology (Scanning Electron Microscopy - SEM), and the quantification of Nitric Oxide (NO) and Reactive Oxygen Species (ROS). The data were analyzed using Kruskal–Wallis and Dunn’s tests (p < 0.05). ResultsThe irradiated groups showed larger viable cells number than the non-irradiated group with LPS (p < 0.0001). All irradiation parameters decreased ROS concentrations after LPS application compared to the non-irradiated group (p < 0.05). All irradiation parameters enhanced the NO values compared to those of the control group (p < 0.05). The SEM images showed cells with regular morphology that adhered to the substrate. ConclusionsAccording to the parameters used in this study, the radiant exposure of 15 J/cm2 and irradiance of 40 mW/cm2 were the most effective irradiation parameters to stimulate and modulate oxidative stress in the primary teeth-derived dental pulp cells.

Sperm characteristics, peroxidation lipid and antioxidant enzyme activity changes in milt of Brycon orbignyanus cryopreserved with melatonin in different freezing curves

The objective of this study was to evaluate extender supplemented with melatonin and freezing curves on the antioxidant enzyme activity, peroxidation lipid and sperm characteristics of cryopreserved Brycon orbignyanus milt. Males (n = 16) and females (n = 5) were hormonally induced with two doses (0.5 mg and 5.0 mg kg−1) of carp pituitary extract, and their gametes were collected by light abdominal massage. The fresh milt was diluted at a ratio of 1:4 (milt:extender) in the following solutions: (Control) 10% methyl glycol (MG) + 5% Beltsville thawing solution; (M1) Control + 1 mM melatonin; and (M2) Control + 2 mM melatonin. The freezing curves were C1 (automated freezer) and C2 (dry shipper for 24 h). After each curve was recorded, the straws were transferred to a liquid nitrogen container until the analyses were performed. The samples were thawed in a water bath (60 °C, 8 s) and evaluated using the Sperm Class Analyzer software for the parameters total motility, progressive motility, curvilinear velocity, straight line velocity, mean displacement velocity, straightness, and linearity. The following were also measured: motility time, vitality, morphology, oxidative stress (lipid peroxidation, activity of catalase and superoxide dismutase, quantification of nitric oxide), and fertilization and hatching rates. The data were analyzed within R by one-way analysis of variance and Tukey's test for comparison of means (p < 0.05). A significant difference (p < 0.05) was observed between the solutions in vitality, morphology, motility, and fertilization rate, the solutions with melatonin having the best values. Total motility, progressive motility, and motility time were significantly different. Among oxidative stress markers, only lipid peroxidation and superoxide dismutase activity showed an effect of the curve × solution interaction (p < 0.05), the solutions with melatonin yielding the lowest values. The fertilization and hatching rates were also higher under the melatonin treatments, regardless of the curve. Melatonin 2 mM and slow curve are indicated for the cryopreservation of fish species sperm as it led to the slowest detrimental spermatozoa effects and better fertilization and hatching rates.

Assessing Biocompatibility of Face Mask Materials during COVID-19 Pandemic by a Rapid Multi-Assays Strategy.

During the coronavirus disease 2019 (COVID-19) pandemic, scientific authorities strongly suggested the use of face masks (FMs). FM materials (FMMs) have to satisfy the medical device biocompatibility requirements as indicated in the technical standard EN ISO 10993-1:2018. The biologic evaluation must be confirmed by in vivo tests to verify cytotoxicity, sensitisation, and skin irritation. Some of these tests require an extensive period of time for their execution, which is incompatible with an emergency situation. In this study, we propose to verify the safety of FMMs combining the assessment of 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT) with quantification of nitric oxide (NO) and interleukin-6 (IL-6), as predictive markers of skin sensitisation or irritation based on human primary fibroblasts. Two hundred and forty-two FMMs were collected and classified according to spectrometer IR in polypropylene, paper, cotton, polyester, polyethylene terephthalate, 3-dimensional printing, and viscose. Of all FMMs tested, 50.8% passed all the assays, 48% failed at least one, and only 1.2% failed all. By a low cost, rapid and highly sensitive multi assays strategy tested on human skin fibroblasts against a large variety of FMMs, we propose a strategy to promptly evaluate biocompatibility in wearable materials.

Immune and nutritional responses of Podisus nigrispinus (Hemiptera: Pentatomidae) nymphs sprayed with azadirachtin

AbstractAzadirachtin is an efficient biopesticide, and it is considered safe for natural enemies because its mode of action is mainly by ingestion. The predator, Podisus nigrispinus Dallas, is found in various agroecosystems as a potential tool for biological control. However, there is much debate on the safety of this stinkbug if azadirachtin is used. Thus, we evaluated the cellular and humoral responses as well as biochemical parameters of the fifth‐instar nymphs of P. nigrispinus treated with azadirachtin. The insects were divided into three treatment groups and sprayed with azadirachtin (Azamax®), deltamethrin (Decis®), and water (control). After 24 h, total and differential hemocyte counts were performed. The analysis of phenoloxidase activity and quantification of nitric oxide, lipids, sugar, glycogen, and protein were performed after an interval of 24 and 72 h. Azadirachtin was found to affect the immune system of this stinkbug and decrease the number of hemocytes, especially pro‐hemocytes and oenocytoids, and it may have caused a reduction in phenoloxidase activity and nitric oxide level. In addition, the decrease in protein levels indicated the interference of azadirachtin in protein synthesis. We conclude that damage caused by the botanical insecticide has been less harmful than that caused by the synthetic insecticide, but use of azadirachtin may still interfere with biological control due to undesirable immune system damage reducing predator efficiency.

New Analogs of Phenacetin with Anti‐Inflammatory Activity

Phenacetin is a compound with pharmacological activity used in the past with antipyretic and analgesic effects. Three new analogues were synthetized aiming improvements in efficacy and less adverse effects, named 4‐ethoxyaniline, R‐71 and R‐72. Nociception and inflammation have been assessed by the formalin‐induced licking response and carrageenan‐induced cell migration models, respectively. Male Swiss webster mice (22–30 g, n = 6–8) were used for both tests, following animal ethics guidelines. The protocol for animal use was approved by CEUA/UFRJ and received the number DFBCICB015‐04/16. Results are compared with vehicle‐treated group and expressed as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p&lt;0.05). Formalin‐induced licking response. The stimulus is given by the injection of 20 μL of formalin 2.5% in mice's left hind paw. The time the animal spends licking the injected paw is recorded. The animals received oral treatment of 4‐ethoxyaniline, R‐71 or R‐72 at 0.1, 1 or 10 mg/kg, acetylsalicylic acid (200 mg/kg) or morphine (2.5 mg/kg) 60 minutes before formalin injection. To access cell migration, the subcutaneous air pouch (SAP) was produced with a sterile air injection and 6 days later, animals received oral treatment with 4‐ethoxyaniline, R‐71 or R‐72 (0.1, 1 or 10 mg/kg) one hour before the injection of sterile carrageenan (1%) or saline into the SAP. After 24 h, all animals were euthanized, the SAP was washed with 1 mL of saline and the exudate was collected for mediators and protein measurements to investigate mechanism of action. Treatment with the three compounds significantly reduced licking time in neurogenic and inflammatory phases of the licking model. 1st phase: vehicle= 52.7 ± 11.3 seconds (sec); 4‐ethoxyaniline: 23.0 ± 5.5* sec; 24.8 ± 10.2* sec; 24.9 ± 3.5* sec; R‐71: 28.8 ± 6.6* sec; 24.2 ± 7.2* sec; 10.2 ± 2.9* sec; R‐72: 16.7 ± 5.6 * sec; 21.7 ± 7.0 * sec; 10.1 ± 3.8 * sec. 2nd phase: vehicle = 212.2 ± 56.3 sec; 4‐ethoxyaniline: 113.8 ± 11.0* sec; 118.2 ± 45.9* sec; 107.8 ± 32.2* sec; R‐71: 126.9 ± 33.3* sec; 137.8 ± 16.2* sec; 82.3 ± 15.9* sec; R72: 129.5 ± 19.6* sec; 109.7 ± 16.2* sec; 60.4 ± 16.6* sec, respectively at doses of 0.1, 1 and 10 mg/kg. Regarding cell migration, 4‐ethoxyaniline reduced 67%, 79% and 77%; R‐71: 66%, 70% and 71%; R‐72: 67%, 69% and 75% when compared to vehicle that also received carrageenan in the pouch. Protein levels were reduced in 4‐ethoxyaniline 31%, 48% and 64%; R‐71: 3%, 15% and 78%; R‐72: 9%, 28% and 70%. NO measurement of 4‐ethoxyaniline reduced 48%, 39% and 3%; R‐71: 56%, 23% and 18%; R‐72: 56%, 44% and 1%. Levels of IL‐1β were also reduced, 4‐ethoxyaniline: 49%, 41% and 55%; R‐71: 38%, 34% and 63%; R‐72: 59%, 16% and 71% respectively at doses of 0.1, 1 and 10 mg/kg. We can conclude that the analogues presented anti‐inflammatory effects since reduced cell migration, NO production and IL‐1β secretion and could be considered to further assays.Support or Funding InformationFinancial Support: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil/Finance Code 001, CNPq and FAPERJ. Leukocyte migration, protein extravasation, Nitric oxide quantification and IL‐1β measurement of treatment with 4‐ethoxyaniline, R‐71 and R‐72. Leukocyte Migration Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 93.4 ± 33.9 93.4 ± 33.9 93.4 ± 33.9 Dexamethasone 31.0* ± 5,5 31.0* ± 5,5 31.0* ± 5,5 4‐Ethoxyaniline 31.6* ± 5.0 20.4* ± 4.7 22.2* ± 6.9 R‐71 32.1* ±4.8 28.9* ± 16.3 27.6* ± 15.3 R‐72 31.7* ± 6.5 29.9* ± 16.6 24.8* ± 8.1 Protein Extravasation Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 703.35 ± 163.9 703.35±163.9 703.35±163.9 Dexamethasone 453.59*±214.5 453.59* ± 214.5 453.59* ± 214.5 4‐Ethoxyaniline 460.8* ± 84.6 345.2*± 181.1 240.8* ± 45.9 R‐71 646.0 ± 147.9 564.7 ± 94.96 214.2* ± 24.6 R‐72 609.5 ± 158.1 483.4 ± 137.3 198.3* ± 28.9 NO Quantification Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 248.63 ± 64.3 248.63 ± 64.3 248.63 ± 64.3 Dexamethasone 112.3* ± 34.9 112.3* ± 34.9 112.3* ± 34.9 4‐Ethoxyaniline 128.3* ± 59.5 149.7 ± 22.1 241.0 ± 86.0 R‐71 109.4* ± 33.48 190.3 ± 74.75 202.3 ± 101.3 R‐72 108.5* ± 36.4 138.2 ± 42.2 244.4 ± 34.9 Levels IL‐1β Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 4016.2 ± 130.6 4016.2 ± 130.6 4016.2 ± 130.6 Dexamethasone 1109.8* ± 307.0 1109.8* ± 307.0 1109.8* ± 307.0 4‐Ethoxyaniline 2038.2 ± 1480.7 2354.9 ± 779.4 1786.7 ± 437.6 R‐71 2472.8 ± 1308.1 2622.4 ± 753.4 1470.5* ± 671.6 R‐72 1637.7 ± 805 3356.6 ± 246.9 1158.8* ± 419.4 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantification of Nitric Oxide and Reactive Oxygen Species in Leishmania-infected J774A.1 Macrophages as a Response to the in vitro Treatment with a Natural Product Compound

Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various mechanisms to evade the immune system and multiply inside the host. Among the main factors of the immunity that are recruited to confront the Leishmania infection are the macrophages (MΦs) that produce effector molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS). Therefore, efficient drug agents should combine the antileishmanial effect of these gaseous transmitters along with the enhancement of the host's adaptive immunity. In the quest of therapeutic alternatives, natural products have been extensively studied and are considered as candidate antileishmanial agents since they exhibit specific properties in modulating the host's immune response towards an effective anti-leishmanial cell-mediated immunity capable to eliminate parasitic dissemination. In the current protocol, Leishmania-infected MΦs (J774A.1 cell line) that have been treated with various increasing concentrations of a natural compound, are tested for the production of the aforementioned molecules. In order to detect NO production, we employ the Griess colorimetric nitrite assay and quantification relies on the construction of an accurate standard curve using appropriate standards of known concentration. ROS detection and quantification is achieved by flow cytometry and relies on the use of carboxy-H2DCFDA, an indicator that converts to a fluorescent form when interacts with ROS molecules.

Free radical stress induces DNA damage response in RAW264.7 macrophages during Mycobacterium smegmatis infection

Genomic instability resulting from oxidative stress responses may be traced to chromosomal aberration. Oxidative stress suggests an imbalance between the systemic manifestation of reactive free radicals and biological system's ability to repair resulting DNA damage and chromosomal aberration. Bacterial infection associated insult is considered as one of the major factors leading to such stress conditions. To study free radical responses by host cells, RAW 264.7 macrophages were infected with non-pathogenic M. smegmatis mc2155 at different time points. The infection process was followed up with an assessment of free radical stress, cytokine, toll-like receptors (TLRs) and the resulting DNA damage profiles. Results of CFU count showed that maximum infection in macrophages was achieved after 9h of infection. Host responses to the infection across different time periods were validated from nitric oxide quantification and expression of iNOS and were plotted at regular intervals. IL-10 and TNF-α expression profile at protein and mRNA level showed a heightened pro-inflammatory response by host macrophages to combat M. smegmatis infection. The expression of TLR4, a receptor for recognition of mycobacteria, in infected macrophages reached the highest level at 9h of infection. Furthermore, comet tail length, micronuclei and γ-H2AX foci recorded the highest level at 9h of infection, pointing to the fact that breakage in DNA double strands in macrophage reaches its peak at 9h of infection. In contrast, treatment with ROS inhibitor N-acetyl-L-cysteine (NAC) prevented host cell death through reduction in oxidative stress and DNA damage response during M. smegmatis infection. Therefore, it can be concluded that enhanced oxidative stress response in M. smegmatis infected macrophages might be correlated with DNA damage response.

Quantification of nitric oxide by high-performance liquid chromatography-fluorometric method in subgenomic hepatitis C virus-replicon expressing Huh7 cells upon treatment with acetylsalicylic acid.

As nitric oxide (NO) expression levels are lower in hepatocytes compared with other cell types, it is difficult to quantify this compound via Griess assay. The aim of the present study was to quantify NO concentration in the cell culture medium from a subgenomic hepatitis C virus (HCV)-replicon expressing Huh-7 cell system using a high-performance liquid chromatography (HPLC)-fluorescence detector in the presence or absence of acetylsalicylic acid (ASA) treatment. HCV-replicon cells were incubated with ASA (4 mM) for 24, 48 and 72 h. Thereafter, the medium was collected to measure nitrites (NO2-) as an indirect indicator of NO levels using diaminonaphtalene as a derivate agent. NO levels were significantly higher (1.7-fold) in Huh-7 replicon cells treated with ASA (72 h post-treatment) than untreated cells (P<0.05); NO inhibitor reduced ~30% the level of NO in Huh-7 replicon cells treated with ASA (48 h post-treatment; P<0.05). The findings suggested that the HPLC-fluorescence method provided an accurate and efficient measurement of NO production in Huh-7-HCV-replicon cells culture medium.

Antitubercular and anti-inflammatory properties screening of natural products from Plectranthus species.

Confirm the use ofPlectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents. Compounds previously isolated fromPlectranthusspp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages withM. tuberculosisH37Rv, using CFU counts assay, at their minimum inhibitory concentration values.Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1×105CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0×105CFU/mL) and isoniazid (1.2×105CFU/mL). The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

Quantification of Nitric oxide in Multiple Myeloma Algerian Patients Using R&amp;D and Arbor Assays Kits

Nitric oxide (NO) is an instable free radical considered as a marker of physiological and pathological processes. NO quantification is very sensitive and shows a great variability because of the NO short life time. Myeloma bone diseases (MBD) constitute one of the most visible aspects in MM cancer, which is a favorable microenvironment for the promotion of communication between malignant plasma cells and other cell types using cellular messengers including NO. On the other hand, many researchers have confirmed the association of osteoporosis with a high concentration of (NO), which has been proven to play a primary role in bone remodeling. The relationship between plasma NO concentration and MM pathology is poorly documented. This work aimed to study the variability of NO quantification using R&amp;D and Arbor assays kits in plasma MM patients in comparison with osteoporosis (OP) patients as positive control. MM and OP blood samples were obtained from patients in three hospitals at the East of Algeria. The results showed a slight similarity (21.56%) between NOx concentrations assayed with two kits, that the average concentration of NOx in MM patients(54, 22μM / l ±20, 06μM/l) was higher than the average concentration of NOx in patients with OP (48,34μM/l ±16,71μM /l). Our results confirmed an important difference and variability in NO quantification Thus the high NOx level production in MM patients comparing with OPpatients was noticed.

Supplementation with l-glutathione improves oxidative status and reduces protein nitration in myenteric neurons in the jejunum in diabetic Rattus norvegicus

Diabetes mellitus is a syndrome with multiple etiologies, characterized by chronic hyperglycemia that increases the production of reactive oxygen species and decreases antioxidant defenses. The present study evaluated oxidative stress parameters and protein nitration in myenteric neurons in the jejunum in diabetic rats supplemented with l-glutathione. Rats (90 days of age) were distributed into four groups (n = 6/group): normoglycemic (N), normoglycemic supplemented with l-glutathione (NGT), diabetic (D), and diabetic supplemented with l-glutathione (DGT). At 210 days of age, the animals were sacrificed, and the jejunum was collected, washed, and subjected to various procedures: tert-butyl hydroperoxide chemiluminescence (CL), determination of total antioxidant capacity (TAC), determination of catalase activity, quantification of nitric oxide (NO), and double-labeling of HuC/D-immunoreactive myenteric neurons and nitrotyrosine (3-NT). Diabetes increased oxidative stress in the jejunum in the D group, reflected by increases in lipid peroxidation, TAC, catalase activity, and NO. The D group exhibited an increase in the percentage of myenteric neurons that were double-labeled with 3-NT. Supplementation with l-glutathione did not cause differences in the average CL curves between the D and DGT groups, but reductions of TAC and catalase activity were observed. Supplementation with l-glutathione promoted a reduction of neurons that contained 3-NT in the DGT group. Diabetes mellitus promoted oxidative stress in the jejunum, and supplementation with l-glutathione improved oxidative status by preventing protein nitration in myenteric neurons in diabetic animals that received supplementation.

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System.

The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Detecting and monitoring NO, SNO and nitrite in vivo.

The detection and quantification of nitric oxide and related reactive nitrogen species in vivo is vital to the understanding of the pathology and/or treatment of numerous conditions. To that end, several detection and quantification methods have been developed to study NO, as well as its redox relatives, nitrite and S-nitrosothiols. While no single technique can offer a complete picture of the nitrogen cycle in a given system in vivo, familiarity with the benefits and limitations of several common tools for NOx determination can assist in the development of new diagnostics and therapeutics.

An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.

NO Fluorescence Quantification by Chitosan CdSe Quantum Dots Nanocomposites

The quantification of nitric oxide (NO) based on the quenching of the fluorescence of a nanocomposites sensor constituted by cadmium/selenium quantum dots (CdSe) stabilized by chitosan (CS) and mercaptosuccinic acid (MSA) is assessed. The optimization of the response of the CS-CdSe-MSA nanocomposites to NO was done by multivariate response surface experimental design methodologies. The highest fluorescence quenching was obtained at pH5.5 and at room temperature. The NO quantification capability of CS-CdSe-MSA was evaluated using standard solutions and a NO donor reagent. A large linear working range from 5 to 200μM and a limit of detection of 1.86μM were obtained. Better quantification results were obtained using the NO donor reagent. Besides NO, the response of the fluorescence of CS-CdSe-MSA to the main reactive oxygen and nitrogen species and similar NO compounds was also assessed.