Characterization and oxygen binding properties of des-Arg human hemoglobin

The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141α could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the α-chain C-terminal residue Arg141α, is a possible model for studies of these interactions. The loss of Arg141α and all the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141α may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Relationship between work rate and oxygen uptake in mitochondrial myopathy during ramp-incremental exercise

We determined the response characteristics and functional correlates of the dynamic relationship between the rate (Δ) of oxygen consumption ( O2) and the applied power output (work rate = WR) during ramp-incremental exercise in patients with mitochondrial myopathy (MM). Fourteen patients (7 males, age 35.4 ± 10.8 years) with biopsy-proven MM and 10 sedentary controls (6 males, age 29.0 ± 7.8 years) took a ramp-incremental cycle ergometer test for the determination of the O2 on-exercise mean response time (MRT) and the gas exchange threshold (GET). The ΔO2/ΔWR slope was calculated up to GET (S1), above GET (S2) and over the entire linear portion of the response (S T). Knee muscle endurance was measured by isokinetic dynamometry. As expected, peak O2 and muscle performance were lower in patients than controls (P < 0.05). Patients had significantly lower ΔO2/ΔWR than controls, especially the S2 component (6.8 ± 1.5 vs 10.3 ± 0.6 mL·min-1·W-1, respectively; P < 0.001). There were significant relationships between ΔO2/ΔWR (S T) and muscle endurance, MRT-O2, GET and peak O2 in MM patients (P < 0.05). In fact, all patients with ΔO2/ΔWR below 8 mL·min-1·W-1 had severely reduced peak O2 values (<60% predicted). Moreover, patients with higher cardiopulmonary stresses during exercise (e.g., higher Δ ventilation/carbon dioxide output and Δ heart rate/ΔO2) had lower ΔO2/ΔWR (P < 0.05). In conclusion, a readily available, effort-independent index of aerobic dysfunction during dynamic exercise (ΔO2/ΔWR) is typically reduced in patients with MM, being related to increased functional impairment and higher cardiopulmonary stress.

Stability of relative oxygen pulse curve during repeated maximal cardiopulmonary testing in professional soccer players

During cardiopulmonary exercise testing (CPET), stroke volume can be indirectly assessed by O2 pulse profile. However, for a valid interpretation, the stability of this variable over time should be known. The objective was to analyze the stability of the O2 pulse curve relative to body mass in elite athletes. VO2, heart rate (HR), and relative O2 pulse were compared at every 10% of the running time in two maximal CPETs, from 2005 to 2010, of 49 soccer players. Maximal values of VO2 (63.4 ± 0.9 vs 63.5 ± 0.9 mL O2•kg-1•min-1), HR (190 ± 1 vs188 ± 1 bpm) and relative O2 pulse (32.9 ± 0.6 vs 32.6 ± 0.6 mL O2•beat-1•kg-1) were similar for the two CPETs (P > 0.05), while the final treadmill velocity increased from 18.5 ± 0.9 to 18.9 ± 1.0 km/h (P < 0.01). Relative O2 pulse increased linearly and similarly in both evaluations (r² = 0.64 and 0.63) up to 90% of the running time. Between 90 and 100% of the running time, the values were less stable, with up to 50% of the players showing a tendency to a plateau in the relative O2 pulse. In young healthy men in good to excellent aerobic condition, the morphology of the relative O2 pulse curve is consistent up to close to the peak effort for a CPET repeated within a 1-year period. No increase in relative O2pulse at peak effort could represent a physiologic stroke volume limitation in these athletes.

Angiotensin-II-induced reactive oxygen species along the SFO-PVN-RVLM pathway: implications in neurogenic hypertension

Neurogenic hypertension has been the subject of extensive research worldwide. This review is based on the premise that some forms of neurogenic hypertension are caused in part by the formation of angiotensin-II (Ang-II)-induced reactive oxygen species along the subfornical organ-paraventricular nucleus of the hypothalamus-rostral ventrolateral medulla pathway (SFO-PVN-RVLM pathway). We will discuss the recent contribution of our laboratory and others regarding the mechanisms by which neurons in the SFO (an important circumventricular organ) are activated by Ang-II, how the SFO communicates with two other important areas involved in sympathetic activity regulation (PVN and RVLM) and how Ang-II-induced reactive oxygen species participate along the SFO-PVN-RVLM pathway in the pathogenesis of neurogenic hypertension.

Electroretinography in dogs using a fiber electrode prototype

We compared two electroretinography (ERG) electrodes in dogs using ERG standards of the International Society for Clinical Electrophysiology of Vision (ISCEV). Ten healthy Yorkshire terrier dogs (mean age, 2.80 ± 1.42 years; 6 females) weighing 5.20 ± 1.56 kg were evaluated using an ERG system for veterinary use. Dark- and light-adapted ERG responses were recorded using an ERG-Jet electrode and a fiber electrode prototype. The examinations were performed during 2 visits, 3 weeks apart. Both electrodes (ERG-Jet or fiber prototype) were used on each animal and the first eye to be recorded (OD × OS) was selected randomly. Three weeks later the examination was repeated on the same animal switching the type of electrode to be used that day and the first eye to be examined. The magnitude and waveform quality obtained with the two electrode types were similar for all ERG responses. ERG amplitudes and implicit times obtained from dogs using the fiber electrode prototype were comparable to those obtained with the ERG-Jet electrode for rod, maximal rod-cone summed, cone, and 30-Hz flicker responses. The fiber electrode prototype is a low-cost device, available as an alternative instrument for clinical veterinary ERG recording for retinal function assessment.

Association between anaerobic components of the maximal accumulated oxygen deficit and 30-second Wingate test

The purpose of this study was to analyze the relationship between the anaerobic components of the maximal accumulated oxygen deficit (MAOD) and of the 30-second Wingate anaerobic test (30-WAnT). Nine male physical education students performed: a) a maximal incremental exercise test; b) a supramaximal constant workload test to determine the anaerobic components of the MAOD; and c) a 30-WAnT to measure the peak power (PP) and mean power (MP). The fast component of the excess post-exercise oxygen consumption and blood lactate accumulation were measured after the supramaximal constant workload test in order to determine the contributions made by alactic (ALMET) and lactic (LAMET) metabolism. Significant correlations were found between PP and ALMET (r=0.71; P=0.033) and between MP and LAMET(r=0.72; P=0.030). The study results suggested that the anaerobic components of the MAOD and of the 30-WAnT are similarly applicable in the assessment of ALMET and LAMET during high-intensity exercise.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Effects of continuous vs interval exercise training on oxygen uptake efficiency slope in patients with coronary artery disease

The oxygen uptake efficiency slope (OUES) is a submaximal index incorporating cardiovascular, peripheral, and pulmonary factors that determine the ventilatory response to exercise. The purpose of this study was to evaluate the effects of continuous exercise training and interval exercise training on the OUES in patients with coronary artery disease. Thirty-five patients (59.3±1.8 years old; 28 men, 7 women) with coronary artery disease were randomly divided into two groups: continuous exercise training (n=18) and interval exercise training (n=17). All patients performed graded exercise tests with respiratory gas analysis before and 3 months after the exercise-training program to determine ventilatory anaerobic threshold (VAT), respiratory compensation point, and peak oxygen consumption (peak VO2). The OUES was assessed based on data from the second minute of exercise until exhaustion by calculating the slope of the linear relation between oxygen uptake and the logarithm of total ventilation. After the interventions, both groups showed increased aerobic fitness (P<0.05). In addition, both the continuous exercise and interval exercise training groups demonstrated an increase in OUES (P<0.05). Significant associations were observed in both groups: 1) continuous exercise training (OUES and peak VO2 r=0.57; OUES and VO2 VAT r=0.57); 2) interval exercise training (OUES and peak VO2 r=0.80; OUES and VO2 VAT r=0.67). Continuous and interval exercise training resulted in a similar increase in OUES among patients with coronary artery disease. These findings suggest that improvements in OUES among CAD patients after aerobic exercise training may be dependent on peripheral and central mechanisms.

Correlation, by multivariate statistical analysis, between the scavenging capacity against reactive oxygen species and the bioactive compounds from frozen fruit pulps

The contents of total phenolic compounds (TPC), total flavonoids (TF), and ascorbic acid (AA) of 18 frozen fruit pulps and their scavenging capacities against peroxyl radical (ROO•), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) were determined. Principal Component Analysis (PCA) showed that TPC (total phenolic compounds) and AA (ascorbic acid) presented positive correlation with the scavenging capacity against ROO•, and TF (total flavonoids) showed positive correlation with the scavenging capacity against •OH and ROO• However, the scavenging capacity against H2O2 presented low correlation with TF (total flavonoids), TPC (total phenolic compounds), and AA (ascorbic acid). The Hierarchical Cluster Analysis (HCA) allowed the classification of the fruit pulps into three groups: one group was formed by the açai pulp with high TF, total flavonoids, content (134.02 mg CE/100 g pulp) and the highest scavenging capacity against ROO•, •OH and H2O2; the second group was formed by the acerola pulp with high TPC, total phenolic compounds, (658.40 mg GAE/100 g pulp) and AA , ascorbic acid, (506.27 mg/100 g pulp) contents; and the third group was formed by pineapple, cacao, caja, cashew-apple, coconut, cupuaçu, guava, orange, lemon, mango, passion fruit, watermelon, pitanga, tamarind, tangerine, and umbu pulps, which could not be separated considering only the contents of bioactive compounds and the scavenging properties.

Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.

Life without Phd? – Phd Oxygen Sensor Proteins in Hypoxic Carcinoma Cell Survival

The microenvironment within the tumor plays a central role in cellular signaling. Rapidly proliferating cancer cells need building blocks for structures as well as nutrients and oxygen for energy production. In normal tissue, the vasculature effectively transports oxygen, nutrient and waste products, and maintains physiological pH. Within a tumor however, the vasculature is rarely sufficient for the needs of tumor cells. This causes the tumor to suffer from lack of oxygen (hypoxia) and nutrients as well as acidification, as the glycolytic end product lactate is accumulated. Cancer cells harbor mutations enabling survival in the rough microenvironment. One of the best characterized mutations is the inactivation of the von Hippel-Lindau protein (pVHL) in clear cell renal cell carcinoma (ccRCC). Inactivation causes constitutive activation of hypoxia-inducible factor HIF which is an important survival factor regulating glycolysis, neovascularization and apoptosis. HIFs are normally regulated by HIF prolyl hydroxylases (PHDs), which in the presence of oxygen target HIF α-subunit to ubiquitination by pVHL and degradation by proteasomes. In my thesis work, I studied the role of PHDs in the survival of carcinoma cells in hypoxia. My work revealed an essential role of PHD1 and PHD3 in cell cycle regulation through two cyclin-dependent kinase inhibitors (CKIs) p21 and p27. Depletion of PHD1 or PHD3 caused a cell cycle arrest and subjected the carcinoma cells to stress and impaired the survival.

Mechanisms of endothelin-1 induced reactive oxygen species production in vascular adventitial fibroblasts

With the relationship between endothelin-1 (ET-1) stimulation and reactive oxygen species (ROS) production unknown in adventitial fibroblasts, I examined the ROS response to ET-1 and angiotensin (Ang II). ET-1 -induced ROS peaked following 4 hrs of ET-1 stimulation and was inhibited by an ETA receptor antagonist (BQ 123, 1 uM) an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (PD98059, 10 uM), and by both a specific, apocynin (10 uM), and non-specific, diphenyleneiodonium (10 uM), NAD(P)H oxidase inhibitor. NOX2 knockout fibroblasts did not produce an ET-1 induced change in ROS levels. Ang II treatment increased ROS levels in a biphasic manner, with the second peak occurring 6 hrs following stimulation. The secondary phase of Ang II induced ROS was inhibited by an ATi receptor antagonist, Losartan (100 uM) and BQ 123. In conclusion, ET-1 induces ROS production primarily through an ETA-ERKl/2 NOX2 pathway, additionally, Ang II-induced ROS production also involves an ETa pathway.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).