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Biomedical subjects

Andrew J Gow

Publications and source records attributed to Andrew J Gow.

At least 19 recordsLinked to original sources
PubMed2006

SP-D-deficient mice are resistant to hyperoxia.

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D-/-) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D-/- mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D-/- mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 +/- 1.4 x 10(5) in SP-D-/- vs. 4.04 +/- 0.25 x 10(5) in SP-D+/+ mice) and neutrophils (1.2 +/- 0.4 x 10(5) vs. 0.03 +/- 0.02 x 10(5) in SP-D-/- and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D-/- mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D-/- mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D-/- phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.

Animals↗
PubMed2006

Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection.

Malaria parasite infection in anopheline mosquitoes is limited by inflammatory levels of nitric oxide metabolites. To assess the mechanisms of parasite stasis or toxicity, we investigated the biochemistry of these metabolites within the blood-filled mosquito midgut. Our data indicate that nitrates, but not nitrites, are elevated in the Plasmodium-infected midgut. Although levels of S-nitrosothiols do not change with infection, blood proteins are S-nitrosylated after ingestion by the mosquito. In addition, photolyzable nitric oxide, which can be attributed to metal nitrosyls, is elevated after infection and, based on the abundance of hemoglobin, likely includes heme iron nitrosyl. The persistence of oxyhemoglobin throughout blood digestion and changes in hemoglobin conformation in response to infection suggest that hemoglobin catalyzes the synthesis of nitric oxide metabolites in a reducing environment. Provision of urate, a potent reductant and scavenger of oxidants and nitrating agents, as a dietary supplement to mosquitoes increased parasite infection levels relative to allantoin-fed controls, suggesting that nitrosative and/or oxidative stresses negatively impact developing parasites. Collectively, our results reveal a unique role for nitric oxide in an oxyhemoglobin-rich environment. In contrast to facilitating oxygen delivery by hemoglobin in the mammalian vasculature, nitric oxide synthesis in the blood-filled mosquito midgut drives the formation of toxic metabolites that limit parasite development.

Allantoin↗
PubMed2006

Biochemical fates of alpha hemoglobin bound to alpha hemoglobin-stabilizing protein AHSP.

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free alpha hemoglobin (alphaHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different alphaHb.AHSP complexes. Binding of AHSP to Fe(II) alphaHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of betaHb to either Fe(II) or Fe(III) alphaHb.AHSP displaces AHSP to generate tetrameric (alpha(2)beta(2)) HbA species. The biochemical properties and in vivo significance of the two alphaHb.AHSP complexes are poorly understood. Here we show that Fe(III) alphaHb.AHSP forms from auto-oxidation of oxygenated alphaHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) alphaHb hemichromes, AHSP-bound Fe(III) alphaHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound alphaHb, either prior to or after extraction by beta subunits. In contrast, reaction of Fe(II) alphaHb-AHSP with betaHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize alphaHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free alphaHb into a more chemically inert state from which recovery of alphaHb is possible by redox cycling.

Binding Sites↗
PubMed2006

Total nitrogen oxide following exercise testing reflects endothelial function and discriminates health status.

Nitric oxide (NO) bioavailability is important in vascular health, but unsuitable as a clinical measure due to biological oxidation. Total nitrogen oxides (NO(x)) are stable but background nitrate levels make it difficult to detect disease-based variation. We investigated the clinical discriminatory value of NO(x) as it relates to exercise capability (VO(2peak)) and brachial artery reactivity (BAR, an NO-dependent measure of endothelial health), in healthy (H), increased risk (RF), and known cardiovascular disease (CVD) subjects. BAR was measured using forearm occlusion/hyperemia stimulus. Subjects performed a maximal graded exercise test (GXT). Blood at rest, exercise termination, and 10 min into recovery was mixed equally with 0.1 M NaOH at 4 degrees C, filtered, and stored at -70 degrees C. NO(x) was measured by chemiluminescence. Seven of the RF group then exercise-trained for 6 months prior to retesting. The H group (n = 12) was younger, had higher VO(2peak), HDL levels, and baseline NO(x) values than the RF (n = 15) and CVD (n = 10) groups. NO(x) increased from baseline to recovery in the H group only (75.85 +/- 19.04 microM vs 97.76 +/- 31.93 microM; P <or= 0.01). BAR was greater in the H versus CVD group (7.24 +/- 3.78% vs 2.59 +/- 3.53%; P <or= 0.01). The relation between VO(2peak) and NO(x) recovery was significant across groups (r = 0.71, P <or= 0.01). Following training the RF subjects (n = 7) increased VO(2peak) (29.98 +/- 6.74 to 33.08 +/- 5.57 ml kg(-1) min(-1); P <or= 0.05), BAR (3.19 +/- 3.96 to 6.86 +/- 4.81%; P <or= 0.05), and recovery NO(x) (18.29 +/- 6.46 to 66.48 + 21.11 microM; P <or= 0.01). These findings suggest that plasma NO(x) following GXT discriminate cardiovascular disease status, are related to regional endothelial function, and respond favorably to exercise training.

Adult↗
PubMed2006

Hormonal regulation of alveolarization: structure-function correlation.

BACKGROUND: Dexamethasone (Dex) limits and all-trans-retinoic acid (RA) promotes alveolarization. While structural changes resulting from such hormonal exposures are known, their functional consequences are unclear. METHODS: Neonatal rats were treated with Dex and/or RA during the first two weeks of life or were given RA after previous exposure to Dex. Morphology was assessed by light microscopy and radial alveolar counts. Function was evaluated by plethysmography at d13, pressure volume curves at d30, and exercise swim testing and arterial blood gases at both d15 and d30. RESULTS: Dex-treated animals had simplified lung architecture without secondary septation. Animals given RA alone had smaller, more numerous alveoli. Concomitant treatment with Dex + RA prevented the Dex-induced changes in septation. While the results of exposure to Dex + RA were sustained, the effects of RA alone were reversed two weeks after treatment was stopped. At d13, Dex-treated animals had increased lung volume, respiratory rate, tidal volume, and minute ventilation. On d15, both RA- and Dex-treated animals had hypercarbia and low arterial pH. By d30, the RA-treated animals resolved this respiratory acidosis, but Dex-treated animals continued to demonstrate blood gas and lung volume abnormalities. Concomitant RA treatment improved respiratory acidosis, but failed to normalize Dex-induced changes in pulmonary function and lung volumes. No differences in exercise tolerance were noted at either d15 or d30. RA treatment after the period of alveolarization also corrected the effects of earlier Dex exposure, but the structural changes due to RA alone were again lost two weeks after treatment. CONCLUSION: We conclude that both RA- and corticosteroid-treatments are associated with respiratory acidosis at d15. While RA alone-induced changes in structure andrespiratory function are reversed, Dex-treated animals continue to demonstrate increased respiratory rate, minute ventilation, tidal and total lung volumes at d30. Concomitant treatment with Dex + RA prevents decreased septation induced by Dex alone and results in correction of hypercarbia. However, these animals continue to have abnormal pulmonary function and lung volumes. Increased septation as a result of RA treatment alone is reversed upon discontinuation of treatment. These data suggest that Dex + RA treatment results in improved gas exchange likely secondary to normalized septation.

Acidosis, Respiratory↗
PubMed2006

The biological chemistry of nitric oxide as it pertains to the extrapulmonary effects of inhaled nitric oxide.

The chemical properties of nitric oxide (NO) have been studied for over 200 years. However, it is only within the last 20 years that the biological implications of this chemistry have been considered. The classical model of NO action within the vasculature centers on production in the endothelium, diffusion to the smooth muscle, and subsequent activation of guanylate cyclase via binding to its heme iron. In the context of this model, it is difficult to conceptualize extrapulmonary effects of inhaled NO. However, NO possesses complex redox chemistry and is capable of forming a range of nitrogen oxide species and is therefore capable of interacting with a variety of biomolecules. Of particular interest is its reaction with reduced cysteine to form an S-nitrosothiol (SNO). SNOs are formed throughout NO biology and are a post-translational modification that has been shown to regulate many proteins under physiologic conditions. Hemoglobin, which was considered to be solely a consumer of NO, can form SNO in a conformationally dependent manner, which allows for the transport of inhaled NO beyond the realm of the lung. Higher oxides of nitrogen are capable of modifying proteins via nitration of tyrosines, which has been shown to occur under pathologic conditions. By virtue of its redox reactivity, one can appreciate that inhaled NO has a variety of routes by which it can act and that these routes may lead to extrapulmonary effects.

Administration, Inhalation↗
PubMed2005

A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension.

The mechanism by which hypoxia [low partial pressure of O(2) (pO(2))] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO(2-)governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). S-nitrosothiol (SNO)-deficient RBCs produce impaired vasodilator responses in vitro and exaggerated pulmonary vasoconstrictor responses in vivo and are defective in oxygenating the blood. RBCs from hypoxemic patients with elevated pulmonary arterial pressure (PAP) exhibit a similar FeNO/SNO imbalance and are thus deficient in pO(2)-coupled vasoregulation. Chemical restoration of SNO-Hb levels in both animals and patients restores the vasodilator activity of RBCs, and this activity is associated with improved oxygenation and lower PAPs.

Animals↗
PubMed2005

Alveolar surfactant protein D content modulates bleomycin-induced lung injury.

RATIONALE: Surfactant protein D (SP-D) is a collectin family member with demonstrated immunomodulatory properties in vitro. We hypothesized that SP-D modulates inflammation during noninfectious lung injury in vivo. OBJECTIVES: To investigate the association of alveolar SP-D and injury, we studied the responses of transgenic mice expressing varying levels of SP-D to intratracheal bleomycin (ITB). METHODS: Eight-week old C57/BL6 SP-D-deficient (-/-) mice and syngeneic wild-type (WT) controls or Swiss Black SP-D-overexpressing (SP-D OE) mice and littermate controls received either ITB or saline and were followed for up to 21 d. MEASUREMENTS AND RESULTS: Kaplan-Meier analysis demonstrated a dose-dependent decrease in survival in ITB SP-D (-/-) mice receiving 2 U/kg bleomycin, with a 14-d mortality of 100% versus 0% mortality for WT receiving 2 U/kg ITB or SP-D (-/-) mice given saline (p < 0.05). At 8 d, ITB SP-D (-/-) mice had greater respiratory distress (frequency/tidal volume) and weight loss than ITB WT mice. Furthermore, bronchoalveolar lavage cellularity, pulmonary parenchymal inflammation, and tissue 3-nitrotyrosine (NO2 Y) were increased to a greater extent in ITB SP-D (-/-) mice. By 21 d, compared with all groups, ITB SP-D (-/-) survivors had increased Trichrome staining and tissue hydroxyproline levels. As proof of principle, SP-D OE mice were highly resistant to bleomycin-induced morbidity and mortality at doses up to 3 U/kg. CONCLUSIONS: These data provide new in vivo evidence for an antiinflammatory role for SP-D in response to noninfectious, subacute lung injury via modulation of oxidative-nitrative stress.

Animals↗
PubMed2005

Structure of oxidized alpha-haemoglobin bound to AHSP reveals a protective mechanism for haem.

The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual alpha- and beta-subunits. The alpha-haemoglobin-stabilizing protein (AHSP) binds alpha-haemoglobin (alphaHb), inhibits the ability of alphaHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous alphaHb is thought to represent a transitional complex through which alphaHb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric alphaHb-AHSP complex at 2.4 A resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of alphaHb undergo drastic structural rearrangements, including the repositioning of several alpha-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from alphaHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes alphaHb and prevents its damaging effects in cells.

Blood Proteins↗
PubMed2005

Regional and whole-body markers of nitric oxide production following hyperemic stimuli.

The measurement of nitric oxide (NO) bioavailability is of great clinical interest in the assessment of vascular health. However, NO is rapidly oxidized to form nitrite and nitrate and thus its direct detection in biological systems is difficult. Venous plasma nitrite (nM concentrations) has been shown to be a marker of forearm NO production following pharmacological stimulation of the endothelium utilizing acetylcholine (Ach). In the present study, we demonstrate, within 15 apparently healthy subjects (34.1 +/- 7.3 years), that reactive hyperemia of the forearm, a physiological endothelial stimulus, results in a 52.5% increase in mean plasma nitrite concentrations (415 +/- 64.0 to 634 +/- 57.1 nM, P = 0.015). However, plasma nitrite is readily oxidized to nitrate within plasma, and thus its utility as a marker of NO production within the clinical setting may be limited. Alternatively, NOx (predominantly nitrate) is relatively stable in plasma (microM concentrations), but is produced by sources other than the vasculature and has been shown to be unsuitable as a measure of localized NO production. We reasoned that the principle source of NOx generation during exercise is NO production and thus have examined the change in NOx following treadmill exercise stress. In this study, 12 apparently healthy subjects showed an increase (from baseline) in venous NOx at peak effort and during recovery (12 +/- 9.1 and 17 +/- 15.3 microM respectively, P < 0.05). In contrast, 10 subjects with cardiovascular disease showed no significant increases. Additionally, a correlation between VO(2peak) and the change in circulating NOx (r(2) = 0.4585, P < or = 0.01) indicated the subjects who could exercise hardest also produced the most NO.

Aged↗
PubMed2005

Invertebrate hemoglobins and nitric oxide: how heme pocket structure controls reactivity.

Hemoglobins (Hbs), generally defined as 5 or 6 coordinate heme proteins whose primary function is oxygen transport, are now recognized to occur in virtually all phyla of living organisms. Historically, study of their function focused on oxygen as a reversibly bound ligand of the ferrous form of the protein. Other diatomic ligands like carbon monoxide and nitric oxide were considered "non-physiological" but useful probes of structure-function relationships in Hbs. This investigatory landscape changed dramatically in the 1980s when nitric oxide was discovered to activate a heme protein, cyclic guanylate cyclase. Later, its activation was likened to Perutz' description of Hb's allosteric properties being triggered by a ligand-dependent "out-of-plane/into-plane" movement of the heme iron. In 1996, a functional role for nitric oxide in human and mammalian Hbs was demonstrated and since that time, the interest in NO as a physiologically relevant Hb ligand has greatly increased. Concomitantly, non-oxygen binding properties of Hbs have challenged the view that Hbs arose for their oxygen storage and transport properties. In this focused review we discuss some invertebrate Hbs' functionally significant reactions with nitric oxide and how strategic positioning of a few residues in the heme pocket plays an large role in the interplay of diatomic ligands to ferrous and ferric heme iron in these proteins.

Allosteric Site↗
PubMed2005

A method to attenuate pneumoperitoneum-induced reductions in splanchnic blood flow.

OBJECTIVE: To determine if increasing nitric oxide bioactivity by inclusion of ethyl nitrite (ENO) in the insufflation admixture would attenuate pneumoperitoneum-induced decreases in splanchnic perfusion. SUMMARY BACKGROUND DATA: Organ blood flow is reduced during pneumoperitoneum and can contribute to laparoscopy-associated morbidity and mortality. Previous attempts to control such decreases in flow have been ineffective. METHODS: Laser-Doppler flow probes were placed on the liver and right kidney of anesthetized pigs. After a baseline recording period, animals were insufflated to a final intraperitoneal pressure of 15 mm Hg. Group one received CO2 (standard practice), whereas group 2 received CO2 plus 100 ppm ENO. Insufflation was maintained for 60 minutes and then the abdomen was manually deflated; monitoring was continued for another 60 minutes. RESULTS: CO2 insufflation (n = 5) cut liver blood flow in half; liver flow remained at this level throughout the postinsufflation period. Inclusion of 100 ppm ENO (n = 6) attenuated both the acute and prolonged blood flow decreases. Statistical modeling of the data showed that, on average, liver blood flow was 14.3 U/min higher in the ENO pigs compared with the CO2 group (P = 0.0454). In contrast, neither treatment significantly altered kidney blood flow (P = 0.6215). CONCLUSION: The data indicate that ENO can effectively attenuate pneumoperitoneum-induced blood flow decreases within the peritoneal cavity. The result suggests a novel therapeutic method of regulating hemodynamic changes during laparoscopic procedures.

Animals↗
PubMed2005

Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia.

Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.

Animals↗
PubMed2004

Molecular mechanism of AHSP-mediated stabilization of alpha-hemoglobin.

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.

Animals↗
PubMed2004

S-Nitrosohemoglobin: an allosteric mediator of NO group function in mammalian vasculature.

Since the discovery of NO as the endothelium-derived relaxing factor, there has been considerable interest in how NO interacts with hemoglobin (Hb). Numerous investigations have highlighted the possibility that rather than operating as a sink to consume NO, the vasculature can operate as a delivery mechanism for NO. The principal hypothesis proposed to explain this phenomenon is that Hb can transport NO on the conserved cysteine residue beta93 and deliver that NO to the tissues in an allosterically dependent manner. This proposal has been termed the S-Nitrosohemoglobin (SNO-Hb) Hypothesis. This review addresses the experimental evidence that led to development of this hypothesis and examines much of the research that resulted from its generation. Specifically it covers the evidence concerning NO in the vasculature, the SNO-Hb Hypothesis itself, the biochemical and biophysical data relating to NO and Hb interactions, SNO-Hb in human physiology, and alternative vascular forms of NO. Finally a model of NO in the vasculature in which SNO-Hb forms the central core is proposed.

Allosteric Site↗
PubMed2004

Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice.

Surfactant protein-D (SP-D), a member of the "collectin" family, has been shown to play a role in innate immunity through modulation of inflammation and clearance of organisms. The role of SP-D in host defense against Pneumocystis carinii pneumonia was assessed using SP-D knockout (KO) mice. When inoculated with P. carinii, both wild-type (wt) and SP-D KO mice required CD4 cell depletion to develop infection. In CD4 cell-depleted models, 2 weeks after infection with P. carinii, SP-D KO mice developed increased intensity of infection, compared with wt mice, despite higher lung-inflammation scores and increased amounts of alveolar inflammatory cells. The increased inflammation seen in SP-D KO mice was accompanied by increases in lung weight, expression of inducible nitric oxide (NO) synthase, total NO levels, and 3-nitrotyrosine levels in lung tissue. These results indicate that SP-D plays a role in host defense against P. carinii in vivo by modulating clearance of organisms, lung inflammation, and metabolism of NO.

Animals↗