Recent Insights Into Nitrite Signaling Processes in Blood

Nitrite was once thought to be inert in human physiology. However, research over the past few decades has established a link between nitrite and the production of nitric oxide (NO) that is potentiated under hypoxic and acidic conditions. Under this new role nitrite acts as a storage pool for bioavailable NO. The NO so produced is likely to play important roles in decreasing platelet activation, contributing to hypoxic vasodilation and minimizing blood-cell adhesion to endothelial cells. Researchers have proposed multiple mechanisms for nitrite reduction in the blood. However, NO production in blood must somehow overcome rapid scavenging by hemoglobin in order to be effective. Here we review the role of red blood cell hemoglobin in the reduction of nitrite and present recent research into mechanisms that may allow nitric oxide and other reactive nitrogen signaling species to escape the red blood cell

Erythrocytes and Vascular Function: Oxygen and Nitric Oxide

Erythrocytes regulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide (NO). First, hemoglobin inside the red blood cell binds oxygen in the lungs and delivers it to tissues throughout the body in an allosterically regulated process, modulated by oxygen, carbon dioxide and proton concentrations. The vasculature responds to low oxygen tensions through vasodilation, further recruiting blood flow and oxygen carrying erythrocytes. Research has shown multiple mechanisms are at play in this classical hypoxic vasodilatory response, with a potential role of red cell derived vasodilatory molecules, such as nitrite derived nitric oxide and red blood cell ATP, considered in the last 20 years. According to these hypotheses, red blood cells release vasodilatory molecules under low oxygen pressures. Candidate molecules released by erythrocytes and responsible for hypoxic vasodilation are nitric oxide, adenosine triphosphate and S-nitrosothiols. Our research group has characterized the biochemistry and physiological effects of the electron and proton transfer reactions from hemoglobin and other ferrous heme globins with nitrite to form NO. In addition to NO generation from nitrite during deoxygenation, hemoglobin has a high affinity for NO. Scavenging of NO by hemoglobin can cause vasoconstriction, which is greatly enhanced by cell free hemoglobin outside of the red cell. Therefore, compartmentalization of hemoglobin inside red blood cells and localization of red blood cells in the blood stream are important for healthy vascular function. Conditions where erythrocyte lysis leads to cell free hemoglobin or where erythrocytes adhere to the endothelium can result in hypertension and vaso constriction. These studies support a model where hemoglobin serves as an oxido-reductase, inhibiting NO and promoting higher vessel tone when oxygenated and reducing nitrite to form NO and vasodilate when deoxygenated. How erythrocytes modulate vascular tone has been widely studied over the last two decades. The vasodilation of the vasculature under hypoxic conditions has inspired much research ranging from the effect of oxygen partial pressure on smooth muscle cell contractility and endothelial nitric oxide synthase (eNOS) activity to nitrite reduction by hemoglobin (Hb) inside erythrocytes and subsequent production of nitric oxide. Here we review how red blood cells (RBCs) and hemoglobin regulate vascular function and blood flow

Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties

Fibrin fibers form the structural backbone of blood clots. The structural properties of fibrin clots are highly dependent on formation kinetics. Environmental factors such as protein concentration, pH, salt, and protein modification, to name a few, can affect fiber kinetics through altered fibrinopeptide release, monomer association, and/or lateral aggregation. The objective of our study was to determine the effect of thrombin and fibrinogen exposed to nitric oxide on fibrin clot properties. ProliNONOate (5 [mu]mol/l) was added to fibrinogen and thrombin before clot initiation and immediately following the addition of thrombin to the fibrinogen solution. Resulting fibrin fibers were probed with an atomic force microscope to determine their diameter and extensibility and fibrin clots were analyzed for clot density using confocal microscopy. Fiber diameters were also determined by confocal microscopy and the rate of clot formation was recorded using UV-vis spectrophotometry. Protein oxidation and S-nitrosation was determined by UV-vis, ELISA, and chemiluminescence. The addition of ProliNONOate to fibrinogen or thrombin resulted in a change in clot structure. ProliNONOate exposure produced clots with lower fiber density, thicker fibers, and increased time to maximum turbidity. The effect of the exposure of nitric oxide to thrombin and fibrinogen were measured independently and indicated that each plays a role in altering clot properties. We detected thrombin S-nitrosation and protein carbonyl formation after nitric oxide exposure. Our study reveals a regulation of fibrin clot properties by nitric oxide exposure and suggests a role of peroxynitrite in oxidative modifications of the proteins. These results relate nitric oxide bioavailability and oxidative stress to altered clot properties

Haptoglobin improves shock, lung injury, and survival in canine pneumonia

During the last half-century, numerous antiinflammatory agents were tested in dozens of clinical trials and have proven ineffective for treating septic shock. The observation in multiple studies that cell-free hemoglobin (CFH) levels are elevated during clinical sepsis and that the degree of increase correlates with higher mortality suggests an alternative approach. Human haptoglobin binds CFH with high affinity and, therefore, can potentially reduce iron availability and oxidative activity. CFH levels are elevated over approximately 24-48 hours in our antibiotic-treated canine model of S. aureus pneumonia that simulates the cardiovascular abnormalities of human septic shock. In this 96-hour model, resuscitative treatments, mechanical ventilation, sedation, and continuous care are translatable to management in human intensive care units. We found, in this S. aureus pneumonia model inducing septic shock, that commercial human haptoglobin concentrate infusions over 48-hours bind canine CFH, increase CFH clearance, and lower circulating iron. Over the 96-hour study, this treatment was associated with an improved metabolic profile (pH, lactate), less lung injury, reversal of shock, and increased survival. Haptoglobin binding compartmentalized CFH to the intravascular space. This observation, in combination with increasing CFHs clearance, reduced available iron as a potential source of bacterial nutrition while decreasing the ability for CFH and iron to cause extravascular oxidative tissue injury. In contrast, haptoglobin therapy had no measurable antiinflammatory effect on elevations in proinflammatory C-reactive protein and cytokine levels. Haptoglobin therapy enhances normal host defense mechanisms in contrast to previously studied antiinflammatory sepsis therapies, making it a biologically plausible novel approach to treat septic shock

Transverse phase-locking in fully frustrated Josephson junction arrays: a new type of fractional giant steps

We study, analytically and numerically, phase locking of driven vortex lattices in fully-frustrated Josephson junction arrays at zero temperature. We consider the case when an ac current is applied {\it perpendicular} to a dc current. We observe phase locking, steps in the current-voltage characteristics, with a dependence on external ac-drive amplitude and frequency qualitatively different from the Shapiro steps, observed when the ac and dc currents are applied in parallel. Further, the critical current increases with increasing transverse ac-drive amplitude, while it decreases for longitudinal ac-drive. The critical current and the phase-locked current step width, increase quadratically with (small) amplitudes of the ac-drive. For larger amplitudes of the transverse ac-signal, we find windows where the critical current is hysteretic, and windows where phase locking is suppressed due to dynamical instabilities. We characterize the dynamical states around the phase-locking interference condition in the $IV$ curve with voltage noise, Lyapunov exponents and Poincar\'e sections. We find that zero temperature phase-locking behavior in large fully frustrated arrays is well described by an effective four plaquette model.Comment: 12 pages, 11 figure

Kaluza-Klein Induced Gravity Inflation

A D-dimensional induced gravity theory is studied carefully in a $4 + (D-4)$ dimensional Friedmann-Robertson-Walker space-time. We try to extract information of the symmetry breaking potential in search of an inflationary solution with non-expanding internal-space. We find that the induced gravity model imposes strong constraints on the form of symmetry breaking potential in order to generate an acceptable inflationary universe. These constraints are analyzed carefully in this paper.Comment: 10 pages, title changed, corrected some typos, two additional comments adde

Measurement of the ttbar Production Cross Section in ppbar Collisions at sqrt{s}=1.96 TeV using Lepton + Jets Events with Secondary Vertex b-tagging

We present a measurement of the ttbar production cross section using events with one charged lepton and jets from ppbar collisions at a center-of-mass energy of 1.96 TeV. In these events, heavy flavor quarks from top quark decay are identified with a secondary vertex tagging algorithm. From 162 pb-1 of data collected by the Collider Detector at Fermilab, a total of 48 candidate events are selected, where 13.5 +- 1.8 events are expected from background contributions. We measure a ttbar production cross section of 5.6^{+1.2}_{-1.1} (stat.) ^{+0.9}_{0.6} (syst.) pb.Comment: 28 pages, 20 figures. Published in Physical Review

Natural Selection Affects Multiple Aspects of Genetic Variation at Putatively Neutral Sites across the Human Genome

A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations

IL-21 and IL-6 Are Critical for Different Aspects of B Cell Immunity and Redundantly Induce Optimal Follicular Helper CD4 T Cell (Tfh) Differentiation

Cytokines are important modulators of lymphocytes, and both interleukin-21 (IL-21) and IL-6 have proposed roles in T follicular helper (Tfh) differentiation, and directly act on B cells. Here we investigated the absence of IL-6 alone, IL-21 alone, or the combined lack of IL-6 and IL-21 on Tfh differentiation and the development of B cell immunity in vivo. C57BL/6 or IL-21−/− mice were treated with a neutralizing monoclonal antibody against IL-6 throughout the course of an acute viral infection (lymphocytic choriomeningitis virus, LCMV). The combined absence of IL-6 and IL-21 resulted in reduced Tfh differentiation and reduced Bcl6 protein expression. In addition, we observed that these cytokines had a large impact on antigen-specific B cell responses. IL-6 and IL-21 collaborate in the acute T-dependent antiviral antibody response (90% loss of circulating antiviral IgG in the absence of both cytokines). In contrast, we observed reduced germinal center formation only in the absence of IL-21. Absence of IL-6 had no impact on germinal centers, and combined absence of both IL-21 and IL-6 revealed no synergistic effect on germinal center B cell development. Studying CD4 T cells in vitro, we found that high IL-21 production was not associated with high Bcl6 or CXCR5 expression. TCR stimulation of purified naïve CD4 T cells in the presence of IL-6 also did not result in Tfh differentiation, as determined by Bcl6 or CXCR5 protein expression. Cumulatively, our data indicates that optimal Tfh formation requires IL-21 and IL-6, and that cytokines alone are insufficient to drive Tfh differentiation