Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Inorganic nitrate supplementation and blood flow restricted exercise tolerance in post-menopausal women.

Exercise tolerance appears to benefit most from dietary nitrate (NO3-) supplementation when muscle oxygen (O2) availability is low. Using a double-blind, randomized cross-over design, we tested the hypothesis that acute NO3- supplementation would improve blood flow restricted exercise duration in post-menopausal women, a population with reduced endogenous nitric oxide bioavailability. Thirteen women (57-76 yr) performed rhythmic isometric handgrip contractions (10% MVC, 30 per min) during progressive forearm blood flow restriction (upper arm cuff gradually inflated 20 mmHg each min) on three study visits, with 7-10 days between visits. Approximately one week following the first (familiarization) visit, participants consumed 140 ml of NO3- concentrated (9.7 mmol, 0.6 gm NO3-) or NO3-depleted beetroot juice (placebo) on separate days (≥7 days apart), with handgrip exercise beginning 100 min post-consumption. Handgrip force recordings were analyzed to determine if NO3- supplementation enhanced force development as blood flow restriction progressed. Nitrate supplementation increased plasma NO3- (16.2-fold) and NO2- (4.2-fold) and time to volitional fatigue (61.8 ± 56.5 s longer duration vs. placebo visit; p = 0.03). Nitrate supplementation increased the rate of force development as forearm muscle ischemia progressed (p = 0.023 between 50 and 75% of time to fatigue) with non-significant effects thereafter (p = 0.052). No effects of nitrate supplementation were observed for mean duration of contraction or relaxation rates (all p > 0.150). These results suggest that acute NO3- supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light.

BACKGROUND: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. METHODS AND RESULTS: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. CONCLUSIONS: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study.

Interaction between the Haptoglobin 2 Phenotype and Diabetes Mellitus on Systolic Pulmonary Arterial Pressure and Nitric Oxide Bioavailability in Hemodialysis Patients.

Elevated systolic pulmonary artery pressure (s-PAP, ≥35 mmHg) serves as an independent predictor of mortality in hemodialysis (HD) and diabetic (DM) patients. A polymorphism in the antioxidant Haptoglobin (Hp) gene has been shown to regulate the bioavailability of nitric oxide (NO), a major mediator of pulmonary vascular tone. We therefore set out to test the hypothesis that the Hp polymorphism may be a determinant of developing elevated s-PAP specifically in the DM state due to a decreased bioavailability of NO. To test our hypothesis we Hp typed and performed transthoracic echocardiography on a series of HD patients and stratified them into elevated and normal s-PAP groups and then evaluated whether there was a significant association between the Hp type, elevated s-PAP, and decreased NO bioavailability as defined by low plasma nitrite. We found a statistically significant interaction between the Hp type and DM on the prevalence of elevated s-PAP and lower mean nitrite levels with the combination of elevated s-PAP and low nitrite levels being significantly more prevalent in Hp 2-2 DM individuals. We conclude that the Hp 2 type is associated with elevated s-PAP levels and low plasma nitrite levels in HD patients specifically in the DM state.

Cytochrome c-mediated formation of S-nitrosothiol in cells.

S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.

Human neuroglobin functions as a redox-regulated nitrite reductase.

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

A novel role for cytochrome c: Efficient catalysis of S-nitrosothiol formation.

Although S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of glutathione to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and S-nitrosoglutathione (GSNO). We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled by an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c into the cytosol during apoptosis potentially releases a GSNO synthase activity that could modulate apoptotic signaling.

Subsequent malignancies in children treated for Hodgkin's disease: associations with gender and radiation dose.

PURPOSE: Subsequent malignant neoplasms (SMNs) are a dominant cause of morbidity and mortality in children treated for Hodgkin's disease (HD). We evaluated select demographic and therapeutic factors associated with SMNs, specifically gender and radiation dose. METHODS AND MATERIALS: A total of 930 children treated for HD at five institutions between 1960 and 1990 were studied. Mean age at diagnosis was 13.6 years, and mean follow-up was 16.8 years (maximum, 39.4 years). Treatment included radiation alone (43%), chemotherapy alone (9%), or both (48%). RESULTS: We found that SMNs occurred in 102 (11%) patients, with a 25-year actuarial rate of 19%. With 15,154 patient years of follow-up, only 7.18 cancers were expected (standardized incidence ratio [SIR] = 14.2; absolute excess risk [AER] = 63 cases/10,000 years). The SIR for female subjects, 19.93, was significantly greater than for males, 8.41 (p < 0.0001). After excluding breast cancer, the SIR for female patients was 15.4, still significantly greater than for male patients (p = 0.0012). Increasing radiation dose was associated with an increasing SIR (p = 0.0085). On univariate analysis, an increased risk was associated with female gender, increasing radiation dose, and age at treatment (12-16 years). Using logistic regression, mantle radiation dose increased risk, and this was 2.5-fold for female patients treated with more than 35 Gy primarily because of breast cancer. CONCLUSIONS: Survivors of childhood HD are at risk for SMNs, and this risk is greater for female individuals even after accounting for breast cancer. Although SMNs occur in the absence of radiation therapy, the risk increases with RT dose.

Unilateral and bilateral breast cancer in women surviving pediatric Hodgkin's disease.

PURPOSE: To define demographic and therapeutic associations with the risk of breast cancer in children treated for Hodgkin's disease (HD), particularly the frequency and interval to the development of contralateral breast cancer. METHODS AND MATERIALS: All 398 female patients (<19 years) treated for HD in five institutions during the accrual period were evaluated. Mean follow-up was 16.9 years. The standardized incidence ratio (SIR) was calculated as the ratio of the observed number of cases to the expected number of cases, estimated using age-matched controls from the Surveillance, Epidemiology, and End Results database. RESULTS: A total of 29 women developed breast cancer (25 invasive, 4 ductal carcinoma in situ; SIR, 37.25; 95% confidence interval, 24.96-53.64). Time to diagnosis was 9.4 to 36.1 years. Cumulative incidence was 24% at 30 years. Ten patients (34%) had bilateral disease (9 metachronous, 1 synchronous). The interval to contralateral breast cancer was 12 to 34 months. On univariate analysis, significant variables included stage of HD, mantle radiation dose, pelvic radiation (protective), and follow-up time. On multivariate analysis, early stage and older age at diagnosis of HD (12 years) were significant predictors of secondary breast cancer. CONCLUSIONS: Women surviving pediatric HD were found to have a 37-fold increase in the risk of breast cancer and a high likelihood of rapidly developing bilateral disease. Early-stage HD and age greater than 12 years at diagnosis of HD were independent risk factors. Higher radiation doses may augment risk, and pelvic radiation may be protective. Breast cancer screening methodology and frequency, plus the role of prophylaxis in patients with unilateral disease, require definition.

Chemiluminescent detection of S-nitrosated proteins: comparison of tri-iodide, copper/CO/cysteine, and modified copper/cysteine methods.

The precise quantification of high and low molecular weight S-nitrosothiols (RSNOs) in biological samples is necessary for the study of nitric oxide-dependent posttranslational signal transduction. Several chemiluminescence-based methods are used for the detection of S-nitrosothiols using the nitric oxide analyzer, including the tri-iodide method and the Cu/CO/cysteine (3C) method. Despite the fact that the tri-idodide method is the most widely used and validated methodology, the levels of S-nitrosated hemoglobin (SNO-Hb) and S-nitrosated albumin have been lower than those reported using photolysis coupled to chemiluminescence. This chapter demonstrates that the tri-iodide method and a newly developed modified copper/cysteine (2C) method compare favorably with the 3C method. Our comparisons include physiologically relevant conditions where the ratio of SNO to heme is low and the frequency of nitrosation of a given Hb tetramer is less than 1. In our studies, the tri-iodide method, the 3C method, and the modified 2C method give consistent and reproducible results. These studies suggest that the proper use of any of these methods can be effective in the accurate measurement of S-nitrosothiols in biological samples. Using more than one in combination has the potential to resolve controversies related to the role of hemoglobin in the generation of RSNOs or the role of RSNOs in biology, whether the RSNOs derive from nitrite or other nitrosative pathways.

Lack of allosterically controlled intramolecular transfer of nitric oxide from the heme to cysteine in the beta subunit of hemoglobin.

The SNO-Hb hypothesis holds that heme-bound nitric oxide (NO) present in the beta subunits of T-state hemoglobin (Hb) will be transferred to the beta-93 cysteine upon conversion to R-state Hb, thereby forming SNO-Hb. A deficiency in the ability of Hb to facilitate this intramolecular transfer has recently been purported to play a role in pulmonary hypertension and sickle cell disease. We prepared deoxygenated Hb samples with small amounts of heme-bound NO and then oxygenated the samples. Electron paramagnetic resonance (EPR) spectroscopy was used to (1) determine the concentration of iron nitrosyl Hb (Fe-NO Hb), (2) show that the NO is evenly distributed among alpha and beta subunits, and (3) show that the Hb undergoes a change in its quaternary state (T to R) upon oxygenation. We did not observe a decrease in the concentration of Fe-NO Hb on oxygenation, which is inconsistent with the prediction of the SNO-Hb hypothesis.

Length of stay and mortality associated with febrile neutropenia among children with cancer.

PURPOSE: The aim of this study was to evaluate risk factors for longer length of stay (los) and mortality among hospitalized children with cancer who have febrile neutropenia. METHODS: This study involved analysis of longitudinal data from the University HealthSystem Consortium database from 1995 to 2002. All patients who were 21 years or younger, with diagnostic codes for both neoplastic disease and febrile neutropenia at discharge, were included. RESULTS: A total of 12,446 patients were identified for the study. The los was 5 days or less for 6,799 patients, and greater than 5 days for 5,647 patients. The mortality rate was 3%. On bivariate analysis, race, age, cancer type, and associated complications (bacteremia/sepsis, hypotension, pneumonia, and fungal infections) were significantly associated with longer length of stay and death. On multivariate analysis, age group, race, cancer type (acute myeloid leukemia, multiple cancers v acute lymphoblastic leukemia), and the complication variables were significantly associated with increased risk of longer los and death. Certain types of cancer (Hodgkin's disease, osteosarcoma/Ewing's sarcoma, rhabdomyosarcoma, compared with acute lymphoblastic leukemia) and year of discharge after 1995 were significantly associated with a reduced risk of longer length of stay and/or mortality. CONCLUSION: Race, age group, year of discharge, associated complications, and cancer type were significantly associated with risk of longer los and mortality. These factors may potentially help in identifying high-risk patients who might benefit from targeted antibiotic therapy or prophylactic hematopoietic growth factor support.