Questioning rotary functionality in the bacterial flagellar system and proposing a murburn model for motility.

Bacterial flagellar system (BFS) was the primary example of a purported 'rotary-motor' functionality in a natural assembly. This mandates the translation of a circular motion of components inside into a linear displacement of the cell body outside, which is supposedly orchestrated with the following features of the BFS: (i) A chemical/electrical differential generates proton motive force (pmf, including a trans-membrane potential, TMP), which is electro-mechanically transduced by inward movement of protons via BFS. (ii) Membrane-bound proteins of BFS serve as stators and the slender filament acts as an external propeller, culminating into a hook-rod that pierces the membrane to connect to a 'broader assembly of deterministically movable rotor'. We had disclaimed the purported pmf/TMP-based respiratory/photosynthetic physiology involving Complex V, which was also perceived as a 'rotary machine' earlier. We pointed out that the murburn redox logic was operative therein. We pursue the following similar perspectives in BFS-context: (i) Low probability for the evolutionary attainment of an ordered/synchronized teaming of about two dozen types of proteins (assembled across five-seven distinct phases) towards the singular agendum of rotary motility. (ii) Vital redox activity (not the gambit of pmf/TMP!) powers the molecular and macroscopic activities of cells, including flagella. (iii) Flagellar movement is noted even in ambiances lacking/countering the directionality mandates sought by pmf/TMP. (iv) Structural features of BFS lack component(s) capable of harnessing/achieving pmf/TMP and functional rotation. A viable murburn model for conversion of molecular/biochemical activity into macroscopic/mechanical outcomes is proposed herein for understanding BFS-assisted motility. HIGHLIGHTSThe motor-like functionalism of bacterial flagellar system (BFS) is analyzedProton/Ion-differential based powering of BFS is unviable in bacteriaUncouplers-sponsored effects were misinterpreted, resulting in a detour in BFS researchThese findings mandate new explanation for nano-bio-mechanical movements in BFSA minimalist murburn model for the bacterial flagella-aided movement is proposedCommunicated by Ramaswamy H. Sarma.

The amniotic fluid proteome changes with term labor and informs biomarker discovery in maternal plasma.

The intra-uterine components of labor, namely, myometrial contractility, cervical ripening, and decidua/membrane activation, have been extensively characterized and involve a local pro-inflammatory milieu of cellular and soluble immune mediators. Targeted profiling has demonstrated that such processes extend to the intra-amniotic space, yet unbiased analyses of the proteome of human amniotic fluid during labor are lacking. Herein, we utilized an aptamer-based platform to characterize 1,310 amniotic fluid proteins and found that the proteome undergoes substantial changes with term labor (251 proteins with differential abundance, q < 0.1, and fold change > 1.25). Proteins with increased abundance in labor are enriched for immune and inflammatory processes, consistent with prior reports of labor-associated changes in the intra-uterine space. By integrating the amniotic fluid proteome with previously generated placental-derived single-cell RNA-seq data, we demonstrated the labor-driven upregulation of signatures corresponding to stromal-3 and decidual cells. We also determined that changes in amniotic fluid protein abundance are reflected in the maternal plasma proteome. Collectively, these findings provide novel insights into the amniotic fluid proteome in term labor and support its potential use as a source of biomarkers to distinguish between true and false labor by using maternal blood samples.

Na,K-ATPase: A murzyme facilitating thermodynamic equilibriums at the membrane-interface.

The redox metabolic paradigm of murburn concept advocates that diffusible reactive species (DRS, particularly oxygen-centric radicals) are mainstays of physiology, and not mere pathological manifestations. The murburn purview of cellular function also integrates the essential principles of bioenergetics, thermogenesis, homeostasis, electrophysiology, and coherence. In this context, any enzyme that generates/modulates/utilizes/sustains DRS functionality is called a murzyme. We have demonstrated that several water-soluble (peroxidases, lactate dehydrogenase, hemogoblin, etc.) and membrane-embedded (Complexes I-V in mitochondria, Photosystems I/II in chloroplasts, rhodopsin/transducin in rod cells, etc.) proteins serve as murzymes. The membrane protein of Na,K-ATPase (NKA, also known as sodium-potassium pump) is the focus of this article, owing to its centrality in neuro-cardio-musculo electrophysiology. Herein, via a series of critical queries starting from the geometric/spatio-temporal considerations of diffusion/mass transfer of solutes in cells to an update on structural/distributional features of NKA in diverse cellular systems, and from various mechanistic aspects of ion-transport (thermodynamics, osmoregulation, evolutionary dictates, etc.) to assays/explanations of inhibitory principles like cardiotonic steroids (CTS), we first highlight some unresolved problems in the field. Thereafter, we propose and apply a minimalist murburn model of trans-membrane ion-differentiation by NKA to address the physiological inhibitory effects of trans-dermal peptide, lithium ion, volatile anesthetics, confirmed interfacial DRS + proton modulators like nitrophenolics and unsaturated fatty acid, and the diverse classes of molecules like CTS, arginine, oximes, etc. These explanations find a pan-systemic connectivity with the inhibitions/uncouplings of other membrane proteins in cells.

The amniotic fluid proteome changes with gestational age in normal pregnancy: a cross-sectional study.

The cell-free transcriptome in amniotic fluid (AF) has been shown to be informative of physiologic and pathologic processes in pregnancy; however, the change in AF proteome with gestational age has mostly been studied by targeted approaches. The objective of this study was to describe the gestational age-dependent changes in the AF proteome during normal pregnancy by using an omics platform. The abundance of 1310 proteins was measured on a high-throughput aptamer-based proteomics platform in AF samples collected from women during midtrimester (16-24 weeks of gestation, n = 15) and at term without labor (37-42 weeks of gestation, n = 13). Only pregnancies without obstetrical complications were included in the study. Almost 25% (320) of AF proteins significantly changed in abundance between the midtrimester and term gestation. Of these, 154 (48.1%) proteins increased, and 166 (51.9%) decreased in abundance at term compared to midtrimester. Tissue-specific signatures of the trachea, salivary glands, brain regions, and immune system were increased while those of the gestational tissues (uterus, placenta, and ovary), cardiac myocytes, and fetal liver were decreased at term compared to midtrimester. The changes in AF protein abundance were correlated with those previously reported in the cell-free AF transcriptome. Intersecting gestational age-modulated AF proteins and their corresponding mRNAs previously reported in the maternal blood identified neutrophil-related protein/mRNA pairs that were modulated in the same direction. The first study to utilize an aptamer-based assay to profile the AF proteome modulation with gestational age, it reveals that almost one-quarter of the proteins are modulated as gestation advances, which is more than twice the fraction of altered plasma proteins (~ 10%). The results reported herein have implications for future studies focused on discovering biomarkers to predict, monitor, and diagnose obstetrical diseases.

The amniotic fluid cell-free transcriptome in spontaneous preterm labor.

The amniotic fluid (AF) cell-free RNA was shown to reflect physiological and pathological processes in pregnancy, but its value in the prediction of spontaneous preterm delivery is unknown. Herein we profiled cell-free RNA in AF samples collected from women who underwent transabdominal amniocentesis after an episode of spontaneous preterm labor and subsequently delivered within 24 h (n = 10) or later (n = 28) in gestation. Expression of known placental single-cell RNA-Seq signatures was quantified in AF cell-free RNA and compared between the groups. Random forest models were applied to predict time-to-delivery after amniocentesis. There were 2385 genes differentially expressed in AF samples of women who delivered within 24 h of amniocentesis compared to gestational age-matched samples from women who delivered after 24 h of amniocentesis. Genes with cell-free RNA changes were associated with immune and inflammatory processes related to the onset of labor, and the expression of placental single-cell RNA-Seq signatures of immune cells was increased with imminent delivery. AF transcriptomic prediction models captured these effects and predicted delivery within 24 h of amniocentesis (AUROC = 0.81). These results may inform the development of biomarkers for spontaneous preterm birth.

How does ascorbate improve endothelial dysfunction? - A computational analysis.

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•-) and peroxynitrite (ONOO-) and increasing eNOS activity. The model predicted that Asc at 0.1-100 µM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1-10 mM reduced O2•- and ONOO- levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.

Megalin-targeting liposomes for placental drug delivery.

Every year, complications during pregnancy affect more than 26 million women. Some of those diseases are associated with significant morbidity and mortality, as is the case of preeclampsia, the main cause of maternal deaths globally. The ability to improve the delivery of drugs to the placenta upon administration to the mother may offer new opportunities in the treatment of diseases of pregnancy. The objective of this study was to develop megalin-targeting liposome nanocarriers for placental drug delivery. Megalin is a transmembrane protein involved in clathrin-mediated endocytic processes, and is expressed in the syncytiotrophoblast (SynT), an epithelial layer at maternal-fetal interface. Targeting megalin thus offers an opportunity for the liposomes to hitchhike into the SynT, thus enriching the concentration of any associated therapeutic cargo in the placental tissue. PEGylated (2 KDa) lipids were modified with gentamicin (GM), a substrate to megalin receptors as we have shown in earlier studies, and used to prepare placental-targeting liposomes. The ability of the targeting liposomes to enhance accumulation of a fluorescence probe was assessed in an in vivo placental model - timed-pregnant Balb/c mice at gestational day (GD) 18.5. The targeting liposomes containing 10 mol% GM-modified lipids increased the accumulation of the conjugated fluorescence probe in the placenta with a total accumulation of 2.8% of the initial dose, which corresponds to a 94 fold increase in accumulation compared to the free probe (p < .0001), and 2-4 fold accumulation compared to the non-targeting control liposomes (p < .0001), as measured by both tissue extraction assay and ex vivo imaging. Furthermore, confocal images of placental SynT cross-sections show a 3-fold increase of the targeting liposomes compared with the non-targeting liposomes. The rate and extent of uptake of a fluorescent probe encapsulated within targeting liposomes was also probed in an in vitro model of the human placental barrier (polarized BeWo monolayers) using flow cytometry. Targeting liposomes containing 5 mol% GM-modified lipids enhanced the uptake of the probe by 1.5 fold compared to the non-targeting control. An increase to 10 mol% of the modified lipid resulted in further enhancement in uptake, which was 2 fold greater compared to control. In a competition assay, inhibition of the megalin receptors resulted in a significant reduction in uptake of the fluorescence probe encapsulated in GM-modified liposomes compared to the uptake without free inhibitor (p < .0001), implicating the involvement of megalin receptor in the internalization of the liposomes. Taken together, these results demonstrate that megalin-targeted liposomes may offer an opportunity to enhance the delivery of therapeutics to the placenta for the treatment of diseases of pregnancy.

The role of glutathione and glutathione peroxidase in regulating cellular level of reactive oxygen and nitrogen species.

Glutathione (GSH) and GSH/glutathione peroxidase (GPX) enzyme system is essential for normal intracellular homeostasis and gets disturbed under pathophysiologic conditions including endothelial dysfunction. Overproduction of reactive oxidative species (ROS) and reactive nitrogen species (RNS) including superoxide (O2•-), and the loss of nitric oxide (NO) bioavailability is a characteristic of endothelial dysfunction. The GSH/GPX system play an important role in eliminating ROS/RNS. Studies have provided important information regarding the interactions of ROS/RNS with the GSH/GPX in biological systems; however, it is not clear how this cross talk affect these reactive species and GSH/GPX enzyme system, under physiologic and oxidative/nitrosative stress conditions. In the present study, we developed a detailed endothelial cell kinetic model to understand the relationship amongst the key enzyme systems including GSH, GPX, peroxiredoxin (Prx) and reactive species, such as hydrogen peroxide (H2O2), peroxynitrite (ONOO-), and dinitrogen trioxide (N2O3). Our simulation results showed that the alterations in the generation rates of O2•- and NO led to the formation of a wide range of ROS and RNS. Simulations performed by varying the ratio of O2•- to NO generation rates as well as GSH and GPX concentrations showed that the GPX reducing capacity was dependent on GSH availability, level of oxidative/nitrosative stress, and can be attributed to N2O3 levels, but not to H2O2 and ONOO-. Our results showed that N2O3 mediated switch-like depletion in GSH and the incorporation of Prx had no considerable effect on the ROS/RNS species other than ONOO- and H2O2. The analysis presented in this study will improve our understanding of vascular diseases in which the levels and oxidation states of GSH, GPX and/or Prx are significantly altered and pharmacological interventions show limited benefits.

Compartmentalized profiling of amniotic fluid cytokines in women with preterm labor.

OBJECTIVE: Amniotic fluid cytokines have been implicated in the mechanisms of preterm labor and birth. Cytokines can be packaged within or on the surface of extracellular vesicles. The main aim of this study was to test whether the protein abundance internal to and on the surface of extracellular vesicles changes in the presence of sterile intra-amniotic inflammation and proven intra-amniotic infection in women with preterm labor as compared to the women with preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection. STUDY DESIGN: Women who had an episode of preterm labor and underwent an amniocentesis for the diagnosis of intra-amniotic infection or intra-amniotic inflammation were classified into three groups: 1) preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 2) preterm labor with sterile intra-amniotic inflammation, and 3) preterm labor with intra-amniotic infection. The concentrations of 38 proteins were determined on the extracellular vesicle surface, within the vesicles, and in the soluble fraction of amniotic fluid. RESULTS: 1) Intra-amniotic inflammation, regardless of detected microbes, was associated with an increased abundance of amniotic fluid cytokines on the extracellular vesicle surface, within vesicles, and in the soluble fraction. These changes were most prominent in women with proven intra-amniotic infection. 2) Cytokine changes on the surface of extracellular vesicles were correlated with those determined in the soluble fraction; yet the magnitude of the increase was significantly different between these compartments. 3) The performance of prediction models of early preterm delivery based on measurements on the extracellular vesicle surface was equivalent to those based on the soluble fraction. CONCLUSIONS: Differential packaging of amniotic fluid cytokines in extracellular vesicles during preterm labor with sterile intra-amniotic inflammation or proven intra-amniotic infection is reported herein for the first time. The current study provides insights into the biology of the intra-amniotic fluid ad may aid in the development of biomarkers for obstetrical disease.

Computational analysis of interactions of oxidative stress and tetrahydrobiopterin reveals instability in eNOS coupling.

In cardiovascular and neurovascular diseases, an increase in oxidative stress and endothelial dysfunction has been reported. There is a reduction in tetrahydrobiopterin (BH4), which is a cofactor for the endothelial nitric oxide synthase (eNOS), resulting in eNOS uncoupling. Studies of the enhancement of BH4 availability have reported mixed results for improvement in endothelial dysfunction. Our understanding of the complex interactions of eNOS uncoupling, oxidative stress and BH4 availability is not complete and a quantitative understanding of these interactions is required. In the present study, we developed a computational model for eNOS uncoupling that considers the temporal changes in biopterin ratio in the oxidative stress conditions. Using the model, we studied the effects of cellular oxidative stress (Qsupcell) representing the non-eNOS based oxidative stress sources and BH4 synthesis (QBH4) on eNOS NO production and biopterin ratio (BH4/total biopterins (TBP)). Model results showed that oxidative stress levels from 0.01 to 1nM·s-1 did not affect eNOS NO production and eNOS remained in coupled state. When the Qsupcell increased above 1nM·s-1, the eNOS coupling and NO production transitioned to an oscillatory state. Oxidative stress levels dynamically changed the biopterin ratio. When Qsupcell increased from 1 to 100nM·s-1, the endothelial cell NO production, TBP levels and biopterin ratio reduced significantly from 26.5 to 2nM·s-1, 3.75 to 0.002µM and 0.99 to 0.25, respectively. For an increase in BH4 synthesis, the improvement in NO production rate and BH4 levels were dependent on the extent of cellular oxidative stress. However, a 10-fold increase in QBH4 at higher oxidative stresses did not restore the NO-production rate and the biopterin ratio. Our mechanistic analysis reveals that a combination of enhancing tetrahydrobiopterin level with a reduction in cellular oxidative stress may result in significant improvement in endothelial dysfunction.

Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H2O2 exposure.

H2O2 mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H2O2 exposure and promote H2O2-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H2O2 exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H2O2 treatment. Primary HUVECs were exposed to transient H2O2 exposure and consistent H2O2 exposure. Following H2O2 treatments for 24, 48 and 72 h, we measured O2(-) production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates. Our results showed that the 24 h H2O2 exposure significantly increased O2(-) levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72 h, HUVECs in both treatment conditions were shown to adapt to reduce O2(-) levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72 h. The response of endothelial cells to excess levels of H2O2 involves a complex interaction amongst O2(-) levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H2O2.

Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality.

Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions.

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor.

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO-RBCs interactions in 'competition experiments'. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO-RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC.

BACKGROUND: Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2⁻), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs. METHOD: Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2⁻ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement. RESULTS: We showed that the O2⁻ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC. CONCLUSION: Our findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.

Endothelial NO and O2·â» production rates differentially regulate oxidative, nitroxidative, and nitrosative stress in the microcirculation.

Endothelial dysfunction causes an imbalance in endothelial NO and O2·â» production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O2·â» production rates. Previous experimental and modeling studies examining the role of NO and O2·â» production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O2·â» production on the complex biochemical NO and O2·â» interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O2·â» to NO or NO to O2·â» production rate ratio (QO2·â»/QNO or QNO/QO2·â», respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both QO2·â»/QNO and QNO/QO2·â» ratios at SOD concentrations of 0.1-100 µM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond QO2·â»/QNO and QNO/QO2·â» ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O2·â» production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.

Computational insights into the role of glutathione in oxidative stress.

Excess reactive oxygen species (ROS) generation and oxidative stress in vascular tissue is associated with many diseases. Glutathione (GSH), one of the most abundant low molecular weight non-protein thiols, modulates physiological levels of ROS and is involved in the cell's oxidative stress response. The GSH/GSSG redox couple is commonly used in measuring oxidative stress status. The imbalance of GSH is reported in many disease states including atherosclerosis, cancer, neurodegenerative disease, and aging. The importance of GSH in modulation of intracellular ROS involves both its protective defense against the damaging effects of oxidative stress and its role in facilitating ROS cell signaling. In this paper, we review significant results obtained from mass balance and kinetic reactions based computational and mathematical models of GSH participation in oxidative stress. The focus is on the mediation of ROS and oxidative stress with respect to the antioxidant capacity of the cell. We discuss the role of GSH in the redox state of the cell, maintaining homeostasis through GSH synthesis, scavenging of free radicals, modulating hydrogen peroxide level and interacting with nitric oxide pathways.

Contribution of membrane permeability and unstirred layer diffusion to nitric oxide-red blood cell interaction.

Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra-cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO-RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (P(m)), hematocrit (Hct) and NO-Hb reaction rate constants on NO-RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO-RBC interactions. The extracellular diffusion contributed 70-90% of total transport resistance for P(m)>1 cm s(-1) whereas membrane resistance accounts for 50-75% of total transport resistance for P(m)<0.1 cm s(-1). We propose a narrow P(m) range of 0.21-0.44 cm s(-1) for 10-45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO-RBC interactions.

Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay.

Superoxide (O(2) (-)) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O(2) (-) is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O(2) (-) generation and a O(2) (-) sensitive fluorescent dye dihydroethidium (DHE) for O(2) (-) measurement. To quantify O(2) (-) and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE-O(2) (-) interaction to understand the effect of O(2) (-) self-dismutation and to quantify DHE-O(2) (-) reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O(2) (-) interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10(3) M(-1) s(-1) for DHE-O(2) (-) reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10(5) M(-1) s(-1). In addition, the O(2) (-) self-dismutation has significant effect on DHE-O(2) (-) interaction. A slower reaction rate of DHE with O(2) (-) is more reasonable for O(2) (-) measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O(2) (-). Results suggest that an accurate measurement of O(2) (-) production rate may be difficult due to competitive interference for many factors; however O(2) (-) concentration may be quantified.

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite.

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O(2)(•-)) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO(-)) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O(2)(•-) and ONOO(-) during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O(2)(•-) production and downstream reactions involving NO, O(2)(•-), ONOO(-), H(2)O(2) and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O(2)(•-), ONOO(-) and H(2)O(2) in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O(2)(•-) production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 µM reduces O(2)(•-) concentration by 90% at all [BH(4)]/[TBP] ratios, (iv) SOD reduces ONOO(-) concentration and increases H(2)O(2) concentration during eNOS uncoupling, (v) Catalase can reduce H(2)O(2) concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.