High Levels Of Nitric Oxide Research Articles

Gestational diabetes mellitus was related to ambient air pollutant nitric oxide during early gestation

BackgroundAmbient air pollution has been linked to the risk of gestational diabetes mellitus (GDM). However, evidence of this association is limited, and no study has examined the effects of nitric oxide (NO). ObjectiveThis study investigated the association between air pollution exposure during gestation and GDM. MethodsThe Taiwan Birth Cohort Study database was used to examine the association between the risk of GDM and all routinely monitored air pollutants among 21,248 women who were pregnant during 2004–2005. We further employed a two-pollutant model for confirming the effect of each pollutant on GDM. ResultsAfter the exclusion criteria were applied, 19,606 women were included in the final analysis. Among them, 378 (1.9%) had been diagnosed as having GDM. These women were older and had higher BMIs than the women without GDM. The risks of GDM onset were significantly associated with NO exposure during the first [adjusted OR (aOR): 1.05, 95% confidence interval (CI): 1.02–1.08] and second (aOR: 1.05, 95%CI: 1.02–1.08) trimesters. Under the two-pollutant model, the effect of NO exposure was also significant during the first (aOR: 1.05, 95%CI: 1.02–1.08) and second (aOR: 1.05, 95%CI: 1.02–1.09) trimesters. ConclusionThe results indicated that exposure to higher NO levels during pregnancy increases the risk of GDM.

Strobilurins as growth-promoting compounds: how Stroby regulates Arabidopsis leaf growth.

Strobilurins are an important class of agrochemical fungicides used throughout the world on a wide variety of crops as protection against fungal pathogens. In addition to this protective role, they are reported to also positively influence plant physiology. In this study, we analysed the effect of Stroby® WG, a commercially available fungicide consisting of 50% (w/w) kresoxim-methyl (KM) as active strobilurin compound, on Arabidopsis leaf growth. Treatment of seedlings with Stroby resulted in larger leaves due to an increase in cell number. Transcriptome analysis of Stroby-treated rosettes demonstrated an increased expression of genes involved in redox homeostasis, iron metabolism and sugar transport. Stroby treatment strongly induced the expression of the subgroup Ib basic helix-loop-helix (bHLH) transcription factors, which have a role in iron homeostasis under iron-limiting conditions. Single loss-of-function mutants of three bHLHs and their triple bhlh039, bhlh100 and bhlh101 mutant did not respond to Stroby treatment. Although iron and sucrose content was not affected, nitric oxide (NO) levels and nitrate reductase (NR) activity were significantly increased in Stroby-treated rosettes as compared with control plants. In conclusion, we suggest that the Stroby-mediated effects on growth depend on the increased expression of the subgroup Ib bHLHs and higher NO levels.

Abstract 216: Hdac2 Modulates Atherosclerosis

Objective: We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in vascular endothelial cell protection against the known endothelial injury stimulus Oxidize Low Density Lipoprotein (OxLDL). The goal of current study is to determine the effects of endothelial-specific HDAC2 overexpression on endothelial dysfunction and atherogenesis in vivo . Approach and Results: To study the effect of endothelial HDAC2 overexpression on vascular function and atherogenesis, we generated and characterized an endothelial specific human HDAC2 overexpressing transgenic mouse (HDAC2-Tg) under the control of the tie2 promoter. The founder HDAC2-overexpressing mice appear to have a normal phenotype- normal weight, mean arterial blood pressure (MAP) and pulse wave velocity (PWV). Isolated intact aortas from endothelial-specific overexpressing HDAC2 transgenic mice (HDAC2-Tg) exhibited a healthier vascular function profile with higher nitric oxide levels and less OxLDL-mediated vascular dysfunction. Further, we induced atherogenesis in C57BL6 and HDAC2-Tg mice by injecting adeno-associated viruses (AAV) encoding PCSK9 (D377Y) under the control of a liver-specific promoter by tail vein injection followed by high fat diet regimen for 12 weeks and measured endothelium-dependent vasorelaxation in isolated blood vessels, pulse wave velocity, an index of vascular stiffness and mean arterial blood pressure. HDAC2-Tg exhibited protected endothelial dependent vascular relaxation and no significant changes in MAP and PWV as compared to control mice. Conclusion: HDAC2 is a critical regulator of endothelial function. Overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis.

Favouring NO over H2O2 production will increase Pb tolerance in Prosopis farcta via altered primary metabolism

Reactive oxygen species (ROS) and nitric oxide (NO) are known in triggering defense functions to detoxify heavy metal stresses. To investigate the relevance of ROS production, Pb treatment (400µM) alone and in combination with 400µM sodium ascorbate (Asc: as H2O2 scavenger) were given to hydroponically grown Prosopis farcta seedlings over a time course of 72h. Data presented here indicate that, the low extent of H2O2 due to scavenging by ascorbate, together with high level of NO improved Pb+Asc- treated Prosopis growth. Following the evoked potential of both the signals, significant increases in phenolic acids; caffeic, ferulic and salicylic acid were observed with Pb treatment; which are consistent with observed increase in lignin content and consequently with growth inhibition. In contrast, Pb+Asc treatment induced more flavonoids (quercetin, kaempferol, luteolin), diminished phenolic acids contents and also lignin. Elicited expression rate of phenylalanine ammonia-lyase gene (PAL) and also its enzymatic activity verified the induced phenylpropanoid metabolism by Pb and Pb+Asc treatments. In comparison with Pb stress, Asc+Pb application induced the high expression of arginine decarboxylase gene (ADC), in polyamines biosynthesis pathway, and conducted the N flow towards polyamines and γ-amino butyric acid (GABA). Examining the impact on enzyme activities, catalase, and guaiacol peroxidase; Pb+Asc reduced activity but this increased ascorbate peroxidase, and aconitase activity. Our observations are consistent with conditions favouring NO production and reduced H2O2 can improve Pb tolerance via wide-ranging effects on a primary metabolic network.

Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity.

Probiotics are live microorganisms that have beneficial effects on host health, including extended lifespan, when they are administered or present in adequate quantities. However, the mechanisms by which probiotics stimulate host longevity remain unclear and very poorly understood. In a recent study (Nat. Commun. 8, 14332 (2017) doi: 10.1038/ncomms14332), we used the spore-forming probiotic bacterium Bacillus subtilis and the model organism Caenorhabditis elegans to study the mechanism by which a probiotic bacterium affects host longevity. We found that biofilm-proficient B. subtilis colonized the C. elegans gut and extended the worm lifespan significantly longer than did biofilm-deficient isogenic strains. In addition to biofilm proficiency, the quorum-sensing pentapeptide CSF and nitric oxide (NO) represent the entire B. subtilis repertoire responsible for the extended longevity of C. elegans. B. subtilis grown under biofilm-supporting conditions synthesized higher levels of NO and CSF than under planktonic growth conditions, emphasizing the key role of the biofilm in slowing host aging. Significantly, the prolongevity effect of B. subtilis was primarily due to a downregulation of the insulin-like signaling system that precisely is a key partaker in the healthy longevity of human centenarians. These findings open the possibility to test if the regular consumption of B. subtilis incorporated in foods and beverages could significantly extend human life expectancy and contribute to stop the development of age-related diseases.

Plasma nitric oxide level is correlated with microvascular functions in the peripheral arterial disease.

At present there is no widely accepted biomarker for monitoring of vascular functions. The purpose of this prospective study was to investigate the association of some blood biomarkers with vascular reactivity in patients with peripheral arterial diseases (PAD). A prospective evaluation was made of 3 groups comprising a control group of healthy individuals, and patients with PAD caused by either atherosclerosis or Buerger's disease. Microvascular perfusion was examined using laser Doppler imaging of cutaneous erythrocyte flux after iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP). The correlation of microvascular reactivity with endothelium-related biomarkers was assessed. ACh-induced and SNP-induced vasodilations were significantly diminished in the PAD groups. The plasma nitric oxide (NO) levels of PAD patients were significantly higher than those of the control group, but asymmetric dimethylarginine, total antioxidant capacity and hydrogen sulphide levels were similar. Plasma NO level was negatively correlated with ACh and SNP-stimulated microvascular flow increase, whereas a positive correlation was detected with blood glucose and glycated hemoglobin (HbA1c) levels in all groups. These results indicate that a high plasma level of NO in PAD patients is associated with diminished endothelium-dependent and independent flow increase in the microvascular bed. An excessive amount of NO-induced nitrosative stress in an inflammatory condition that might be a reason for vascular dysfunction should be taken into consideration in the diagnostic and therapeutic approaches to PAD.

A fucan of a brown seaweed and its antitumoral property on HT-29 and immunomodulatory activity in murine RAW 264.7 macrophage cell line

Polysaccharides from algae are also proper candidates with therapeutic properties and immunomodulatory and antitumor effects. The brown seaweed Lobophora variegata synthesizes different groups of anionic polysaccharides with several biological properties. Sulfated polysaccharides were obtained by delipidation of seaweed, proteolysis, and fractionation with different volumes of acetone. A fraction of sulfated polysaccharides, fucans or fucoidans, was extracted with 0.8 v acetone and named L. variegata (LV). This fucan was assessed in the inflammatory process in rats, antitumor action on human colon adenocarcinoma (HT-29 cells), apoptosis, and its effect on the cell cycle. LV fraction, a galactofucan, exhibited a high ratio of total sugar/sulfate (1.5) and a very low level of proteins. This polysaccharide showed an antiinflammatory effect on two models of inflammation induced by croton oil and oxazolone in rats. LV was analyzed in cellular proliferation of HT-29. We also demonstrated the cytotoxic action against this cell line and induction in the apoptosis and decreased the cell cycle in phases S and G 2/M and the accumulation of cells in the G 1 phase. Our studies with LV showed a marked immunomodulatory action without and with lipopolysaccharide (LPS) on RAW 264.7 cells. Comparative studies of two fucans, LV and FV (Fucus vesiculosus), with different structures were assessed on viability in macrophages, RAW 264.7 cells. Both showed cytotoxicity in these cells. We observed high levels of nitric oxide (NO) production, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) when treated at 0.25, 0.5, and 1.0 mg mL−1 of LV. Data also suggest that LV has potential antitumor effects on HT-29 and antiinflammatory and immunomodulatory effects on RAW 264.7 cells.

Potent Inhibition of Nitric Oxide-Releasing Bifendate Derivatives against Drug-Resistant K562/A02 Cells in Vitro and in Vivo.

Multidrug resistance is a major obstacle to successful chemotherapy for leukemia. In this study, a series of nitric oxide (NO)-releasing bifendate derivatives (7a-n) were synthesized. Biological evaluation indicated that the most active compound (7a) produced relatively high levels of NO and significantly inhibited the proliferation of drug-resistant K562/A02 cells in vitro and in vivo. In addition, 7a induced the mitochondrial tyrosine nitration and the intracellular accumulation of rhodamine 123 by inhibiting P-gp activity in K562/A02 cells. Furthermore, 7a remarkably down-regulated AKT, NF-κB, and ERK activation and HIF-1α expression in K562/A02 cells, which are associated with the tumor cell proliferation and drug resistance. Notably, the antitumor effects were dramatically attenuated by an NO scavenger or elimination of the NO-releasing capability of 7a, indicating that NO produced by 7a contributed to, at least partly, its cytotoxicity against drug-resistant K562/A02 cells. Overall, 7a may be a potential agent against drug-resistant myelogenous leukemia.

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-β until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1β until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

Oxidative Stress in the Development of Genetic Generalised Epilepsy: An Observational Study in Southern Indian Population.

Oxidative stress resulting from excessive generation of Reactive Oxygen Species (ROS) plays a significant role in neurodegeneration associated with seizures/epilepsy. To evaluate oxidative stress markers and antioxidant enzymes in Genetic Generalised Epilepsy (GGE) and to know the extent of oxidative stress induced by Anti-Epileptic Drugs (AEDs) with the time duration of treatment. In this case-control study, 310 GGE patients (male:female=203:107), who were on AED treatment (n=235) and 75 untreated patients (male:female=49:26) along with 310 age and sex matched healthy controls were recruited. Oxidative stress markers such as Nitric Oxide (NO), Malondialdehyde (MDA) and antioxidant enzyme activities namely Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx) and Catalase (CAT) were measured spectrophotometrically. Significantly higher levels of serum NO, MDA and low levels of plasma Total Antioxidant Capacity (TAC) were found in patients as compared to controls (p<0.001) whereas erythrocyte SOD, CAT and GPx activities were found to be significantly low in patients when compared to the control group (p<0.001). Statistically significant higher levels of NO, MDA and lower levels of SOD, CAT and TAC were observed in patients subgroup, who were on AEDs for more than >5 years compared to other groups (≤ 1 year and 1-≤ 5 years) (p=0.02, p=0.01, p=0.001, p=0.01 and p=0.05 respectively). Further, significant increase in the levels of NO, MDA and decreased activities of SOD, CAT were found in treated patients compared to untreated patients (p<0.05) denoting that additional oxidative stress induced by AEDs which results in seizure recurrence and drug intractability. Our study demonstrated that GGE patients have additional oxidative stress due to AEDs and decreased antioxidant enzyme activities causing an imbalance between oxidant and antioxidant status, which might contribute to the pathogenesis of GGE.

RISK FACTORS AND MARKERS OF TUBULOINTERSTITIAL NEPHRITIS DEVELOPMENT IN CHILDREN WITH OXALATE-CALCIUM CRYSTALLURIA

Research objective: to study the clinical and biochemical risk factors and markers for the formation of tubulointerstitial nephritis in children with oxalate-calcium crystalluria. Methods: 30 children with tubulointerstitial nephritis were examined on a background of oxalate-calcium crystalluria. Special clinical and laboratory methods of research were used: stable metabolites of nitric oxide-nitrites and activity of superoxide dismutase, albumin in urine were determined in erythrocytes and urine. To determine the clinical features of the course and identify risk factors for the development of tubulointerstitial nephritis, we analyzed: perinatal and genealogical anamnesis, the age of debut of oxalate-calcium crystalluria and tubulointerstitial nephritis, and ultrasound data from the organs of the urinary system. Results: In children with tubulointerstitial nephritis, in 22% of cases the signs of oxalate-calcium crystal-luria preceded the underlying disease. For tubulointerstitial nephritis, which occurred against the background of metabolic disturbances, whose debut occurred at an early age, a latent course is typical, and minimal tubular kidney dysfunction, formed at the age of 4–11 years. The following clinical and anamnestic factors had the greatest impact on the formation of tubulointerstitial nephritis from a single-factor analysis: a family history with family history and the presence of 2 degrees of kinship of urolithiasis, a threat of termination of pregnancy and gestosis of the first half of pregnancy, hyperechoic inclusions in the renal parenchyma by ultrasound examination of child. In parallel with the processes of antioxidant protection against the background of oxidative stress, there is an increase in the level of metabolites of nitric oxide in the urine, and in combination with albuminuria, an increase in endothelial dysfunction. Conclusion: the leading factors in the formation of tubulointerstitial nephritis in children with oxalate-calcium crystalluria are an aggravated genealogical anamnesis due to the presence of urolithiasis in relatives of the first and second degree of kinship. According to the results of special research methods, albuminuria, low activity of superoxide dismutase in the urine, higher levels of nitric oxide in erythrocytes and urine indicate the progression of tubulointerstitial nephritis and are markers of the inflammatory process.

Biomarkers of Oxidative Stress in HIV Seropositive Individuals on Highly Active Antiretroviral Therapy

Oxidative and nitrosative stress have been implicated in the pathogenesis and progression of human immune deficiency virus (HIV) infection, and seem to be more pronounced with commencement of highly active antiretroviral therapy (HAART). Evaluation of markers of oxidative and nitrosative stress may be useful indices for monitoring progress of infection and development of adverse drug reactions. The total antioxidant capacity (TAC), total plasma peroxides (TPP), oxidative stress index (OSI), nitric oxide (NO), reduced form of glutathione (GSH), malondialdehyde (MDA), and catalase activity were estimated in HIV-infected individuals with or without HAART. A total of 90 consenting subjects comprising of 30 HIV seropositive subjects on HAART, 30 HAART naive and 30 apparently healthy HIV seronegative controls resident in Calabar were recruited into the study. TAC, TPP, NO, GSH, MDA, and catalase activity were determined using colorimetric methods, CD4+ T cell count was done by flow cytometry while body mass index (BMI) and oxidative stress index were determined by calculation. Data was analyzed using ANOVA, LSD post hoc and Pearson’s correlation at p<0.05. The results showed that HIV seronegative controls had higher CD4+ T cell count, BMI, NO, and catalase activity and lower TPP compared to HIV seropositive individuals on HAART and HAART naive subjects (p < 0.05). Higher levels of NO and catalase and lower CD4+ T cells count, MDA, TPP, and OSI were observed in HIV on HAART compared to HAART naive (p < 0.05). OSI correlated positively with TPP (r = 0.547, p = 0.002) and negatively with TAC (r = ‒0.727, p = 0.000) in HIV HAART naive subjects only. In conclusion, HAART may be associated with reduction in lipid peroxidation and restoration of some antioxidants, and their levels may determine initiation of HAART in HIV-infected individuals.

Hyperglycemia and high nitric oxide level induced oxidative stress in the brain and molecular alteration in the neurons and glial cells of laboratory mouse, Mus musculus

Chronic hyperglycemia (glucotoxicity) is reported to have detrimental effects on various brain functions leading to neurodegenerative changes. However, the effect of hyperglycemia in combination with high nitric oxide (NO) level (reported to be increased during glucotoxicity), on brain functions is not clear yet. The present study was designed to investigate the effects of hyperglycemic drug Streptozotocin (STZ) and NO donor Sodium nitroprusside (SNP) on the brain of laboratory mouse, Mus musculus. Effects of these conditions were studied on the markers of oxidative stress, NF-κB signalling and the markers of neuronal and glial cell activation/inflammation. Results indicate increased level of MDA and altered antioxidant enzymes activity in both the treated groups compared to control but high levels of AGEs, AOPP and AR activity (markers of diabetic complications) were observed in STZ group only. On the other hand, while STZ group showed decreased IL-6 level, it was increased in SNP group but IFN-ϒ level increased in both the treated groups compared to control. Further, in addition to alterations in the expressions of iNOS, IKKβ, IKBα and NF-κB subunits (RelA-p65/RelB-p50) observed in the neurons and glial cells of different brain regions (hypothalamus, basolateral amygdala and cerebral cortex), enhanced expression of microglial CD11b and astrocytic GFAP was also found in both the treated groups compared to control.Present findings led us to conclude that both hyperglycemia and high NO level causes oxidative stress in addition to molecular alteration in the neurons and glial cells. It is suggested that high blood glucose and NO level induced oxidative stress may lead to neuroinflammation possibly via NF-κB signalling.

Bioactivities of n-hexane fraction of Vateria copallifera and GC–MS analysis of its phytoconstituents

Vateria copallifera (Retz.) is an important timber source belonging to the family of Dipterocarpaceae, which is acknowledged as a plant family rich in a variety of bioactive chemical constituents. This study was conducted to evaluate the chemical composition and the bioactivities of the non-polar n-hexane fraction obtained by dry column chromatography of the hexane extract of V. copallifera bark. n-Hexane fraction was analyzed by gas chromatography–mass spectrometry (GC–MS). Cytotoxicity was measured by the MTT assay against HeLa, MCF7, HepG2, HCT116 and SK-Mel 5 cell lines. The neuroprotective effect of the n-hexane fraction was assessed by oxygen glucose deprivation (OGD) assay using PC12 cells. Nitric oxide (NO) inhibitory activity was investigated on lipopolysaccharide (LPS)-stimulated microglial cells. The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and oxygen radical absorbance capacity (ORAC) assays. Cholinesterase inhibitory activity was evaluated using acetylcholinesterase and butyrylcholinesterase inhibitory assays and protease inhibitory activity was evaluated by α-chymotrypsin and elastase enzyme inhibitory assays. Decane, dodecane, undecanes, naphthalene derivative, heptadecane, squalene, copaene and eicosane were identified as the major constituents (>2% peak area). The n-hexane fraction exhibited a concentration-dependent increase in the neuroprotective activity although it was not significant (p>0.05) at the tested concentrations. Contradictorily, the n-hexane fraction treated microglia cells released significantly (p<0.05) higher levels of NO into the medium (>100%) compared to the control counterpart. However, further studies are needed to assess the neuroprotective activity and to determine the cause of the increase of NO. The n-hexane fraction showed no cytotoxicity, antioxidant, ChE, protease and GST enzyme inhibitory activity at the tested concentrations. To our knowledge, this is the first report on the analysis of the chemical composition and the bioactive potential of the non-polar n-hexane fraction obtained by chromatography of the hexane extract of V. copallifera bark.

Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection

Oxidative stress during peripartum period may compromise the uterine immunity. In the present study, we assessed the oxidative stress and antioxidant status during peripartum period and studied their relationship with postpartum uterine infection in dairy cows. Peripheral blood concentrations of total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) were determined (day −21, −7, on the day of calving and day +7, +21, +35) in normal (n=11), puerperal metritic (n=7) and clinical endometritic (n=6) cows. Endometrial biopsy was performed on the day of calving and expression of CAT, GPx4 and SOD2 genes was studied using qRT-PCR. Puerperal metritic cows had significantly (P<0.05) lower TAC (on day −7, day 0, day +7, +21 & +35), higher MDA (on day −21, −7 & on the day of calving) and NO (on day 0, +7 & day +35) concentrations compared to normal cows. Similarly, clinical endometritic cows had significantly (P<0.05) lower TAC (on day −7, 0, +7 & +21), higher MDA (on day −21, −7, +7 and +35) and NO (on day +7, +21 & +35) concentrations compared to normal cows. The expression of CAT and GPx4 genes was lower (P<0.05) and SOD2 gene was higher (P<0.05) in endometrial tissue of cows that developed uterine infection compared to normal cows. The relationship of peripheral levels of MDA and NO with antioxidant enzymes expression in endometrial tissue was found significant. Receiver operator characteristic analysis revealed that the concentrations of TAC on day −7 to day +35, MDA on day −21 to day +7 and NO on the day of calving to day +35 were highly correlated to the development of postpartum uterine infection in cows. It may be inferred that the low serum TAC level and high level of lipid peroxidation and NO during peripartum period influenced the endometrial expression of anitioxidative genes that compromised the uterine health during postpartum period.

Cardiomyogenesis of periodontal ligament-derived stem cells by dynamic tensile strain.

Cellular therapies for the treatment of myocardial infarction have proven to be an invaluable tool in recent years and provide encouraging evidence for the possibility to restore normal heart function. However, questions still remain as to the optimal cell source, pre-conditioning methods and delivery techniques for such an application. This study explores the use of a population of stem cells arising from the neural crest and isolated from adult human periodontal ligament along with short-term mechanical strain as an inducer of cardiomyogenesis and possibly pre-conditioning stimulus for cellular cardiomyoplasty. Cells were subjected to a short-term dynamic mechanical tension in our custom-built bioreactor and analyzed for cardiomyogenic commitment. Mechanical strain elicited a cardiomyogenic response from the cells following just 2h of stimulation. Mechanical strain activated and translocated cardiac-specific transcription factors GATA4, MEF2C and Nkx2.5, and induced expression of the sarcomeric actin and cardiac troponin T proteins. Mechanical strain induced production of significantly higher levels of nitric oxide when compared to static controls. Elimination of elevated ROS levels by free radical scavengers completely abolished the cardiomyogenic response of the cells. MicroRNA profile changes in stretched cells were detected for 39 miRNAs with 16 of the differentially expressed miRNAs related to heart development. The use of stem cells in combination with mechanical strain prior to their delivery in vivo may pose a valuable alternative for the treatment of myocardial infarction and merits further exploration for its capacity to augment the already observed beneficial effects of cellular therapies.

A photodynamic bifunctional conjugate for prostate cancer: an in vitro mechanistic study.

Photodynamic therapy (PDT) has drawn considerable attention for its efficacy against certain types of cancers. It shows however limits in the case of deep cancers, favoring tumor recurrence under suboptimal conditions. More insight into the molecular mechanisms of PDT-induced cytotoxicity and cytoprotection is essential to extend and strengthen this therapeutic modality. As PDT induces iNOS/NO in both tumor and microenvironment, we examined the role of nitric oxide (NO) in cytotoxicity and cytoprotection. Our findings show that NO mediates its cellular effects by acting on the NF-κB/YY1/RKIP loop, which controls cell growth and apoptosis. The cytoprotective effect of PDT-induced NO is observed at low NO levels, which activate the pro-survival/anti-apoptotic NF-κB and YY1, while inhibiting the anti-survival/pro-apoptotic and metastasis suppressor RKIP. In contrast, high PDT-induced NO levels inhibit NF-κB and YY1 and induce RKIP, resulting in significant anti-tumor activity. These findings reveal a critical role played by NO in PDT and suggest that the use of bifunctional PDT agents composed of a photosensitizer and a NO-donor could enhance the photo-treatment effect. A successful application of NO in anticancer therapy requires control of its concentration in the target tissue. To address this issue we propose as PDT agent, a bimolecular conjugate called DR2, composed of a photosensitizer (Pheophorbide a) and a non-steroidal anti-androgen molecule capable of releasing NO under the exclusive control of light. The mechanism of action of DR2 in prostate cancer cells is reported and discussed.

S-nitroso-N-acetyl penicillamine inhibits spontaneous exit from metaphase-II arrest in rat eggs cultured in vitro.

Present study was designed to investigate the in vitro effects of nitric oxide (NO) donor such as S-nitroso-N-acetyl penicillamine (SNAP) on spontaneous exit from metaphase-II arrest (SEM-II) in rat eggs cultured in vitro. Ovulated eggs were denuded and then exposed to various concentrations (0.0, 0.01, 0.1 and 1.0mM) of SNAP for 3h under in vitro culture conditions. The percentage of SEM-II, specific and total phosphorylated cyclin-dependent kinase-1 (Cdk1), cyclin B1 and anaphase promoting complex/cyclosome (APC/C) levels as well as Cdk1 activity were analyzed. The SEM-II was associated with a decrease of Thr-161 phosphorylated Cdk1 as well as cyclin B1 levels and increase of Thr-14/Tyr-15 phosphorylated Cdk1, APC/C levels and Cdk1 activity in aged eggs cultured in vitro. On the other hand, SNAP treatment prevented a decrease of Thr-161 phosphorylated Cdk1 as well as cyclin B1 levels and increase of Thr-14/Tyr-15 phosphorylated Cdk1, Cdk1 activity that finally prevented SEM-II in a concentration-dependent manner. However, APC/C level was not affected by SNAP during the course of treatment in vitro. Present data suggest that SNAP prevented SEM-II possibly by increasing high level of NO and thereby maturation promoting factor (MPF) stabilization in rat eggs cultured in vitro. Hence, SNAP could be used to prevent SEM-II that reduces reproductive outcome in several mammalian species.

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity and inflammatory regulation. Here, DMBT1 expression was analyzed by immunohistochemistry in postmortem lung sections from patients with MAS. The lung epithelial cell line A549, stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty expression plasmid (DMBT1- cells), was exposed to meconium. NO was determined in dependence of aminoguanidine (inducible NO synthase inhibitor), steroids and lipopolysaccharide (LPS). DMBT1 is highly expressed in lungs with MAS. In the absence of meconium, DMBT1+ cells showed a higher NO production than the DMBT1- cells (p=0.0090). Meconium led in DMBT1- and DMBT1+ cells to elevated NO levels (p<0.0001), but with a higher NO level in DMBT1+ cells (p<0.0001). Aminoguanidine, an iNOS inhibitor, reduced the higher NO production in DMBT1+ cells (p=0.0476), but NO levels remained above NO production from DMBT1- cells (p=0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p=0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p<0.0001). In comparison to exposure with only meconium, the combined addition of LPS and meconium to the cells increased NO levels in both DMBT1- cells (p=0.0030) and DMBT1+ cells (p=0.0028). In conclusion, basal and meconium-induced NO production in lung epithelial cells is positively regulated by DMBT1.

Elevated nitric oxide levels associated with hepatic cell apoptosis during liver injury.

Hepatic injury is a major event in liver surgery such as liver transplantation and it always leads to hepatic cell apoptosis. Nitric oxide (NO) is a key signaling regulation molecule. Many researchers have shown that increased NO level can influence liver cell apoptosis by promoting or inhibiting the relative signaling pathways that are involved in the caspase family, Bax/Bcl-2, mitochondria, oxidative stress, death receptors, and mitogen-activated protein kinases. Elucidating the relationships between NO and hepatic cell apoptosis is necessary for ameliorating prognosis of liver surgery. This article reviews the newest research progress in the relationships between higher NO levels and hepatic cell apoptosis in liver injury.