Systemic Nitric Oxide Production Research Articles

Systemic nitric oxide metabolites and the chance of pre-diabetes regression to normoglycemia: A 9-year cohort study.

We aimed to track longitudinal changes of glycemic status in subjects with pre-diabetes (Pre-DM) in relation to their baseline levels of systemic nitric oxide (NO) production [i.e., measured as serum NO metabolites (NOx), crude and body weight (BW)-adjusted NOx to creatinine ratio (NOx-to-Cr)] over 9 years. This cohort study included 541 middle-aged Iranian men and women with Pre-DM, recruited in 2006-2008 and followed up to 2015-2017. The colorimetric Griess method was used to measure serum NOx concentration. Multinomial logistic regression analyses estimated the odds ratios (OR) of Pre-DM regression and progression across tertiles (tertile 3 vs. tertile 1 and tertile 2) of serum NOx, crude, and BW-adjusted NOx-to-Cr ratio. Participants who regressed to normoglycemia (NG) had a higher BW-adjusted NOx-to-Cr ratio than those who developed type 2 diabetes (T2D) or those who remained Pre-DM (0.52±0.34 vs. 0.43±0.25 and 0.48±0.29, P=0.023). Higher BW-adjusted NOx-to-Cr increased chance of returning to NG (OR=2.05, 95% CI= 0.98-4.32, P=0.058) and decreased levels of 2h-serum glucose over time (P time×group=0.025), as well as the decreased overall mean of fasting (106, 95% CI=103-109 vs. 110, 95% CI=108-112 mg/dL, P=0.008) and 2h-serum glucose (153, 95% CI=146-159 vs. 163, 95% CI=158-168 mg/dL, P=0.018). A higher endogenous NO production (i.e., indirectly measured by BW- and Cr-adjusted serum NOx concentration) in Pre-DM subjects is associated with the chance of returning to NG.

Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization.

The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system.

Plasma and amniotic fluid concentrations of nitric oxide: Effects on uterine artery and placental vasculature in women who underwent voluntary pregnancy termination and in women with missed and threatened abortion. A pilot study

ObjectivesFirst trimester miscarriage is a multifactorial event. Various angiogenic factors have been proposed as possible early markers of non-viable pregnancies. The aim of the present study was to evaluate the systemic nitric oxide (NO) production in healthy early pregnancy and its possible role in first trimester miscarriage. Study designWe prospectively enrolled women referred to our Unit for elective termination of pregnancy, threatened abortion or missed abortion. Blood samples were taken for testing circulating NO plasma levels. Subsequently, all patients underwent 2-D ultrasonographic analysis and Color Doppler imaging to assess the pulsatility index of the uterine arteries. 3-D ultrasonographic and power Doppler analysis allowed a volumetric and vascular reconstruction of the placenta. During dilatation and vacuum aspiration, amniotic fluid was collected. ResultsSeventy-two patients were enrolled: 25 with elective termination of pregnancy (Group I); 17 with threatened abortion (Group II); 30 with missed abortion (Group III). Group II showed greater placental volume and lower uterine arteries PI than others. The plasma NO concentration resulted statistically higher in women with threatened abortion, while amniotic fluid NO concentration were higher in the viable pregnancies (Group I) than in the aborted fetuses (Group III). Plasma NO was inversely correlated with both mean arterial pressure and uterine artery PI and was positively correlated with amniotic fluid NO and CRL; amniotic fluid NO was positively correlated with placental Vascularization Index and Vascularization-Flow Index. ConclusionAmniotic NO concentration was higher in viable pregnancies and positively related to Doppler 3D indices of vascularization and blood flow within the placenta. Further studies are needed to elucidate its role in first trimester miscarriage.

Cardiac Protection by Oral Sodium Thiosulfate in a Rat Model of L-NNA-Induced Heart Disease.

Hypertension contributes to cardiac damage and remodeling. Despite the availability of renin-angiotensin system inhibitors and other antihypertensive therapies, some patients still develop heart failure. Novel therapeutic approaches are required that are effective and without major adverse effects. Sodium Thiosulfate (STS), a reversible oxidation product of hydrogen sulfide (H2S), is a promising pharmacological entity with vasodilator and anti-oxidant potential that is clinically approved for the treatment of calciphylaxis and cyanide poisoning. We hypothesized that Sodium Thiosulfate improves cardiac disease in an experimental hypertension model and sought to investigate its cardioprotective effects by direct comparison to the ACE-inhibitor lisinopril, alone and in combination, using a rat model of chronic nitric oxide (NO) deficiency. Systemic nitric oxide production was inhibited in Sprague Dawley rats by administering N-ω-nitro-l-arginine (L-NNA) with the food for three weeks, leading to progressive hypertension, cardiac dysfunction and remodeling. We observed that STS, orally administered via the drinking water, ameliorated L-NNA-induced heart disease. Treatment with STS for two weeks ameliorated hypertension and improved systolic function, left ventricular hypertrophy, cardiac fibrosis and oxidative stress, without causing metabolic acidosis as is sometimes observed following parenteral administration of this drug. STS and lisinopril had similar protective effects that were not additive when combined. Our findings indicate that oral intervention with a H2S donor such as STS has cardioprotective properties without noticeable side effects.

Acute Soy Supplementation Improves 20-km Time Trial Performance, Power, and Speed.

Isoflavones, a chemical class of phytoestrogens found in soybeans and soy products, may have biological functions similar to estradiol. After binding with ERβ or perhaps independently of estrogen receptors, isoflavones may augment vascular endothelial relaxation, contributing to improved limb blood flow. To determine if acute fermented soy extract supplementation influences 20-km time trial cycling performance and cardiac hemodynamics compared with a placebo. Subjects included 25 cyclists and triathletes (31 ± 8 yr, V˙O2peak: 55.1 ± 8.4 mL·kg·min). Each subject completed a V˙O2peak assessment, familiarization, and two 20-km time trials in randomized order after ingestion of a fermented soy extract supplement or placebo. The fermented soy extract consisted of 30 g powdered supplement in 16 fl. ounces of water. The placebo contained the same quantities of organic cocoa powder and water. Each trial consisted of 60 min of rest, 30 min at 55% Wpeak, and a self-paced 20-km time trial. Soy supplementation elicited a faster time to 20-km completion (-0.22 ± 0.51 min; -13 s), lower average HR (-5 ± 7 bpm), and significantly greater power (7 ± 3 W) and speed (0.42 ± 0.16 km·h) during the last 5 km of the time trial compared with placebo. Analysis of the results by relative fitness level (<57 vs ≥ 57 mL⋅kg⋅min) indicated that those with a higher level of fitness reaped the largest performance improvement alongside a reduced HR (-5 ± 7 bpm). Ingestion of a fermented soy extract supplement improved sprint-distance performance through improvements in both power and speed. For those with great aerobic fitness, soy supplementation may help to decrease cardiac demand alongside performance improvement.

All-trans retinoic acid (ATRA) prevents lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment in aged rats

We aimed to investigate preventive effects of All-trans retinoic acid (ATRA) on a lipopolysaccharide (LPS)-induced aged neuroinflammation model. We analyzed behavior, systemic nitric oxide (NO) production, cerebral NO synthase (NOS2) and β-amyloid (Aβ) 1–42 expression and tissue integrity in the neuroinflammation model pretreated with ATRA (150μg/ml/rat/day) for 30days. Our results showed that LPS treatment (500μg/kg/day) for 7days disturbed memory, enhanced systemic NO production, NOS2 and Aβ 1–42 cerebral expression and generated an Alzheimer's disease (AD)-like neuronal degeneration. Interestingly, ATRA pretreatment prevented the LPS-induced deleterious effects. ATRA could be a potent preventive approach in AD.

Altered Nitric Oxide System in Cardiovascular and Renal Diseases.

Nitric oxide (NO) is synthesized by a family of NO synthases (NOS), including neuronal, inducible, and endothelial NOS (n/i/eNOS). NO-mediated effects can be beneficial or harmful depending on the specific risk factors affecting the disease. In hypertension, the vascular relaxation response to acetylcholine is blunted, and that to direct NO donors is maintained. A reduction in the activity of eNOS is mainly responsible for the elevation of blood pressure, and an abnormal expression of iNOS is likely to be related to the progression of vascular dysfunction. While eNOS/nNOS-derived NO is protective against the development of atherosclerosis, iNOS-derived NO may be proatherogenic. eNOS-derived NO may prevent the progression of myocardial infarction. Myocardial ischemia/reperfusion injury is significantly enhanced in eNOS-deficient animals. An important component of heart failure is the loss of coronary vascular eNOS activity. A pressure-overload may cause severer left ventricular hypertrophy and dysfunction in eNOS null mice than in wild-type mice. iNOS-derived NO has detrimental effects on the myocardium. NO plays an important role in regulating the angiogenesis and slowing the interstitial fibrosis of the obstructed kidney. In unilateral ureteral obstruction, the expression of eNOS was decreased in the affected kidney. In triply n/i/eNOS null mice, nephrogenic diabetes insipidus developed along with reduced aquaporin-2 abundance. In chronic kidney disease model of subtotal-nephrectomized rats, treatment with NOS inhibitors decreased systemic NO production and induced left ventricular systolic dysfunction (renocardiac syndrome).

Systemic Nitric Oxide (NO) Production is Increased in Children with Sickle Cell Disease (SCD) Receiving Fortified Supplementary Food

BackgroundLow bioavailable NO is implicated in the pathophysiology of SCD. Within a clinical trial of a daily ready‐to‐use supplementary food (RUSF) fortified with arginine (ARG, 0.2g/kg/d), the substrate of endothelial NO synthase, and citrulline (CITR, 0.1g/kg/d) vs. non fortified RUSF we conducted a nested feasibility study of systemic NO production.MethodsChildren (N=29, 70% male, 9‐11 years) with SCD underwent a modified Oral Nitrate Test (ONT) with reduced fasting time to measure the decay in serial saliva samples of an oral dose of labelled nitrate (Na15NO3) by GC/MS [figure 1] (Siervo et al. 2011). Both groups received intervention for 12 weeks before the ONT and a further 5 weeks before plasma amino acids (Biochrom 30) and NO mediated endothelial function were assessed by flow mediated dilatation (FMDmax). Data were analysed using Wilcoxan ranksum or signrank tests.imageResultsThe intervention groups were similar for age and weight. NO production (µmol/kg/hr) was not associated with age or sex. Results by intervention group and for both intervention groups combined are presented in [figure 2]. No associations between NO production and amino acid concentrations or endothelial function (FMDmax) were observed.&#x0D;imageConclusionsThis method is feasible in young children in a low resource setting. NO production in malnourished children with SCD can be increased by protein energy supplementation and may be increased further with additional arginine and citrulline.

Systemic production of nitric oxide during severe asthma: immonumodulation by the helminth echinococcus granulosus

Background Several studies highlight the implication of nitric oxide (NO) in the airways inflammation during asthma. Moreover, asthma is thought to be associated with a systemic inflammation. However, little is known about the systemic production of NO in asmathic patients. In this sense, our aim was to evaluate the in vivo and ex vivo production of NO in Algerian patients with severe asthma. Moreover, the effect of helminthes infection on asthma is still inconclusive. In this sense, the second part of this study aims to investigate the effect of the helminth Echinococcus granulosus on this production. This parasite causes cyst formation particularly in lungs and liver.

Nitric Oxide and Hydrogen Peroxide Production are Involved in Systemic Drought Tolerance Induced by 2R,3R-Butanediol in Arabidopsis thaliana.

2R,3R-Butanediol, a volatile compound produced by certain rhizobacteria, is involved in induced drought tolerance in Arabidopsis thaliana through mechanisms involving stomatal closure. In this study, we examined the involvement of nitric oxide and hydrogen peroxide in induced drought tolerance, because these are signaling agents in drought stress responses mediated by abscisic acid (ABA). Fluorescence-based assays showed that systemic nitric oxide and hydrogen peroxide production was induced by 2R,3R-butanediol and correlated with expression of genes encoding nitrate reductase and nitric oxide synthase. Co-treatment of 2R,3R-butanediol with an inhibitor of nitrate reductase or an inhibitor of nitric oxide synthase lowered nitric oxide production and lessened induced drought tolerance. Increases in hydrogen peroxide were negated by co-treatment of 2R,3R-butanediol with inhibitors of NADPH oxidase, or peroxidase. These findings support the volatile 2R,3R-butanediol synthesized by certain rhizobacteria is an active player in induction of drought tolerance through mechanisms involving nitric oxide and hydrogen peroxide production.

Nitric oxide production by endotoxin preparations in TLR4-deficient mice

Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4−/− is not accompanied by hypotension or morbidity.

TLR2 activation causes no morbidity or cardiovascular failure, despite excessive systemic nitric oxide production

Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated.

Suppression of inflammatory cytokines in the kidney during chronic high salt intake in wild type and e‐NOS knockout mice

Inhibition of systemic nitric oxide (NO) production reduces interleukin (IL‐10) and increases tumor necrosis factor‐α (TNF‐α) level in the kidney. However, the status of inflammatory cytokines during chronic high salt (HS) intake, particularly in NO deficient condition, is not yet clearly known. In the present study, we measured the renal levels of pro‐ (TNF‐α and IL‐6) and anti‐ (IL‐10) inflammatory cytokines after HS (4% NaCl for 2 weeks) intake in both wild‐type (WT, C57BL/6) and e‐NOS knockout (KO) mice (~8 weeks old, n=6 in each group) and compared these findings with those in mice with normal salt intake (NS; 0.3% NaCl). The kidneys were collected under anesthesia at the end of experimental period and the cytokine levels were measured in homogenized renal tissues using ELISA kits. Mean arterial pressure (tail‐cuff plethysmography) was increased in KO (111±4 to 136±8 mmHg) but not in WT (99±5 to107±4 mmHg) after HS intake. The renal level of cytokines (expressed in pg/mg protein) during NS intake were significantly higher in KO (TNF‐α, 624±67 vs 325±73; IL‐6, 619±106 vs 166±61; IL‐10, 6087±567 vs 3929±378) compared to those in WT. However, HS intake significantly lowered the levels of these cytokines both in WT (TNF‐α, 114±17; IL‐6, 81±14; IL‐10, 865±130) and in KO (TNF‐α, 115±18; IL‐6, 56±7; IL‐10, 882±141). These data demonstrate that chronic HS intake suppresses both pro‐ and anti‐inflammatory cytokines levels in the kidney in intact as well as in eNOS deficient condition. (NHLBI #66432)

Glucocorticoids downregulate systemic nitric oxide synthesis and counteract overexpression of hepatic heme oxygenase-1 during endotoxin tolerance

Heme oxygenase (HO)-1 has antioxidant and cytoprotective properties if properly expressed, whereas nitric oxide (NO) impairs tissue perfusion when greatly increased in the blood circulation. Here we hypothesized that the NO and HO-1 systems are altered during lipopolysaccharide (LPS) tolerance, and that glucocorticoids are crucial modulators of systemic NO production and hepatic HO-1 expression during this intriguing phenomenon of cellular reprogramming. Adrenalectomized (ADX) rats with or without administration of dexamethasone (DEX) were challenged with LPS for 3 consecutive days. The plasma levels of corticosterone and nitrate (NOx), and expression of HO-1 protein were assessed. During tolerance, corticosterone levels were elevated, NOx reduced, and HO-1 overexpressed. ADX rats challenged with LPS for 3 consecutive days exhibited a ~9-fold increase in NOx and a ~6-fold increase in HO-1, reverted by DEX. Our findings strongly support the fact that glucocorticoids downregulate systemic NO synthesis and counteract hepatic HO-1 overexpression during LPS tolerance.

Effect of cardiopulmonary bypass on tissue injury markers and endothelial activation during coronary artery bypass graft surgery

Coronary artery bypass grafting (CABG) is done either using cardiopulmonary bypass (CPB) or without using CPB (OPCAB). But, recently, reports have shown that CPB is associated with increased postoperative morbidity because of the involvement of many systems. The aim of this prospective study was to evaluate the influence of the technique of surgery on various tissue injury markers and the extent of endothelial activation in patients undergoing CABG and OPCAB coronary revascularization. This study was conducted at a tertiary healthcare center during the period May 2008 to December 2009. This was a prospective nonrandomized blinded study. The activities of Creatine Phosphokinase (CK) and its isoenzyme CK-MB, Lactate dehydrogenase (LDH), levels of cardiac Troponin I, soluble vascular cell adhesion molecule-1 (sVCAM-I) and systemic nitric oxide production were assessed. All the results were expressed as Mean ± SD. P value ≤ 0.05 was considered significant. The statistical analysis was carried out using SPSS Version 11.5-computer software (SPSS Inc., Chicago, IL, USA). The surgical trauma had elevated CK, CK-MB and Troponin I in both the groups and further elevation was seen in the CABG group in comparison to OPCAB (P<0.001). The Troponin I concentrations showed an increase from 0.11 ± 0.02 preoperatively to 6.59 ± 0.59 (ng/ml) at 24 h (P<0.001) compared to the OPCAB group. Mean serum levels of sVCAM-1 increased significantly after surgery in both the groups (P<0.02). To determine serum nitric oxide (NO) production, NO2- and NO3- (stable end products of NO oxidation) were analyzed which also increased significantly at 24 h in both the groups. But the increase was not significant at 48 h in both the groups compared to the preoperative value in our study. The present study indicates that, despite comparable surgical trauma, the OPCAB significantly reduces tissue injury. The overall pattern of endothelial activation after OPCAB is significantly lower than that after CABG. This may contribute to improved organ function, and improved postoperative recovery.

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Effects of chronic leptin administration on nitric oxide production and immune responsiveness of greenfinches

Leptin and nitric oxide (NO) are both important messengers in intra- and intercellular communication systems in vertebrates. Several studies have demonstrated an involvement of both substances in the immune response. Here we tested the effects of chronic leptin and anti-leptin treatments on the NO production and phytohaemagglutinin- (PHA) induced cutaneous inflammatory response in a wild passerine, the greenfinch (Carduelis chloris). Plasma leptin levels of individual birds were consistent in time but could be still temporarily increased by administration of recombinant chicken leptin. Increase of plasma leptin was also induced by administration of anti-leptin, which can be most likely explained by increased endogenous leptin production due to disruption of signalling pathways. Contrary to previous findings in mammals, leptin administration reduced systemic NO production. Leptin increased cutaneous swelling response to PHA. This immune-enhancing effect was observable despite the similar plasma leptin levels of leptin-treated and control birds at the time of measurement of immune responses, i.e., 9days after start of the treatments. This provides evidence for a delayed or long-term potentiation of the cells and cytokines involved. The effects of leptin administration on NO production and immune responsiveness were age-dependent, which indicates the complexity of underlying regulatory mechanisms.

Acetyl salicyclic acid (aspirin) improves synthesis of maspin and lowers incidence of metastasis in breast cancer patients

Maspin, a 42 kDa protein produced in normal breast cells, has been shown to inhibit the invasion and metastasis of breast cancer in an animal model. Ingestion of acetylsalicylic acid (aspirin) by breast cancer patients has been reported to restore the systemic synthesis of maspin through the stimulation of systemic nitric oxide production. Studies were carried out to determine the effect of aspirin on the incidence of breast cancer metastasis, which is reported to occur in 50% of patients who have previously received chemotherapy, radiation, and/or surgery. Thirty-five female patients (aged 41-65 years) with breast cancer who had previously received these therapies took one 75 mg/70 kg body weight enteric-coated aspirin tablet every 24 h, after an adequate meal, for 3 years. Their plasma nitric oxide and maspin levels were measured. The occurrence of metastasis was ascertained monthly by a qualified oncologist, and confirmed, if necessary, by biopsy. Daily ingestion of aspirin by participants resulted in an increase in maspin levels from 0.95 ± 0.04 to 4.63 ± 0.05 nM after 24 h. These levels were maintained for 3 years. These studies suggest that daily ingestion of aspirin might significantly reduce the incidence of breast cancer metastasis in patients who have previously received anticancer therapies.

Local and systemic production of nitric oxide in tomato responses to powdery mildew infection

Various genetic and physiological aspects of resistance of Lycopersicon spp. to Oidium neolycopersici have been reported, but limited information is available on the molecular background of the plant-pathogen interaction. This article reports the changes in nitric oxide (NO) production in three Lycopersicon spp. genotypes which show different levels of resistance to tomato powdery mildew. NO production was determined in plant leaf extracts of L. esculentum cv. Amateur (susceptible), L. chmielewskii (moderately resistant) and L. hirsutum f. glabratum (highly resistant) by the oxyhaemoglobin method during 216 h post-inoculation. A specific, two-phase increase in NO production was observed in the extracts of infected leaves of moderately and highly resistant genotypes. Moreover, transmission of a systemic response throughout the plant was observed as an increase in NO production within tissues of uninoculated leaves. The results suggest that arginine-dependent enzyme activity was probably the main source of NO in tomato tissues, which was inhibited by competitive reversible and irreversible inhibitors of animal NO synthase, but not by a plant nitrate reductase inhibitor. In resistant tomato genotypes, increased NO production was localized in infected tissues by confocal laser scanning microscopy using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. NO production observed in the extracts from pathogen conidia, together with elevated NO production localized in developing pathogen hyphae, demonstrates a complex role of NO in plant-pathogen interactions. Our results are discussed with regard to a possible role of increased NO production in pathogens during pathogenesis, as well as local and systemic plant defence mechanisms.

Chronic ingestion of Porphyromonas gingivalis induces systemic nitric oxide response in mice

Porphyromonas gingivalis induces nitric oxide (NO) production in various cells, systemic NO elevation being expected in chronic oral challenge. Groups of BALB/c mice were inoculated orally with either live P. gingivalis ATCC 33277 or sterile broth on days 0, 2 and 4, with or without later administration of the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Plasma and tissues were harvested on day 42 for assays of tumor necrosis factor-alpha (TNF-alpha), nitrite and nitrate (NOx) and tissue NO, or histology and iNOS immunohistochemistry. No signs of gingivitis were observed, but plasma NOx was significantly elevated (P = 0.028) as was TNF-alpha (P = 0.079) in P. gingivalis-inoculated animals compared with controls, NOx being reduced when 1400W was used. NO production in organs showed a similar trend, with significant elevation in liver (P = 0.017) and kidneys (P = 0.027), whereas concomitant treatment of inoculated animals with 1400W caused significant reductions in NO in aorta (P = 0.008) and kidneys (P = 0.046). Sham-inoculated 1400W-treated animals had significantly increased plasma NOx (P = 0.004) and liver NO (P = 0.04). NOx in plasma correlated significantly with NO production in lungs (0.35, P = 0.032) and kidneys (0.47, P = 0.003). Immunohistochemistry demonstrated iNOS activity in many tissues in all groups. Repeated oral administration of P. gingivalis induced systemic NO and NOx production in mice, probably by activating iNOS as suggested by the response to 1400W.