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1.  Effects of spermine NONOate and ATP on protein aggregation: light scattering evidences 
BMC Biophysics  2013;6:1.
Background and objective
Regulating protein function in the cell by small molecules, provide a rapid, reversible and tunable tool of metabolic control. However, due to its complexity the issue is poorly studied so far. The effects of small solutes on protein behavior can be studied by examining changes of protein secondary structure, in its hydrodynamic radius as well as its thermal aggregation. The study aim was to investigate effects of adenosine-5’-triphosphate (ATP), spermine NONOate (NO donor) as well as sodium/potassium ions on thermal aggregation of albumin and hemoglobin. To follow aggregation of the proteins, their diffusion coefficients were measured by quasi-elastic light scattering (QELS) at constant pH (7.4) in the presence of solutes over a temperature range from 25°C to 80°C.
Results and discussion
1) Spermine NONOate persistently decreased the hemoglobin aggregation temperature Tairrespectively of the Na+/K+ environment, 2) ATP alone had no effect on the protein’s thermal stability but it facilitated protein’s destabilization in the presence of spermine NONOate and 3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions.
The ATP effect on protein aggregation was ambiguous: ATP alone had no effect on the protein’s thermal stability but it facilitated protein’s destabilization in the presence of nitric oxide. The magnitude and direction of the observed effects strongly depended on concentrations of K+ and Na+ in the solution.
PMCID: PMC3561150  PMID: 23289636
2.  Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)α1-specific activity and glucose transport in human skeletal muscle 
Diabetologia  2010;53(6):1142-1150.
We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport.
Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 µmol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined.
In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-α1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 µmol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-α1 phosphorylation.
Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-α1-associated activity.
PMCID: PMC2860569  PMID: 20349036
Contraction; Exercise; GLUT4; Spermine NONOate
3.  Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure 
Nucleic Acids Research  2001;29(24):5121-5128.
The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris–HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.
PMCID: PMC97540  PMID: 11812845
4.  Long-lasting inhibition of presynaptic metabolism and neurotransmitter release by protein S-nitrosylation 
Free radical biology & medicine  2010;49(5):757-769.
Nitric oxide (NO) and related reactive nitrogen species (RNS) play a major role in the pathophysiology of stroke and other neurodegenerative diseases. One of the poorly understood consequences of stroke is a long-lasting inhibition of synaptic transmission. In this study, we tested the hypothesis that RNS can produce long-term inhibition of neurotransmitter release via S-nitrosylation of proteins in presynaptic nerve endings. We examined the effects of exogenous sources of RNS on the vesicular and non-vesicular L-[3H]glutamate release from rat brain synaptosomes. NO/RNS donors, such as spermine NONOate, MAHMA NONOate, S-nitroso-L-cysteine, and SIN-1, inhibited only the vesicular component of glutamate release with the order of potency that closely matched levels of protein S-nitrosylation. Inhibition of glutamate release persisted for >1 hr after RNS donor decomposition and wash out, and strongly correlated with decreases in the intrasynaptosomal ATP levels. Post-NO treatment of synaptosomes with thiol-reducing reagents decreased the total content of S-nitrosylated proteins but had little effect on glutamate release and ATP levels. In contrast, post-NO application of the end-product of glycolysis pyruvate partially rescued neurotransmitter release and ATP production. These data suggest that RNS suppress presynaptic metabolism and neurotransmitter release via poorly reversible modifications of glycolytic and mitochondrial enzymes, one of which was identified as glyceraldehyde-3-phosphate dehydrogenase. Similar mechanism may contribute to the long-term suppression of neuronal communication during nitrosative stress in vivo.
PMCID: PMC2923826  PMID: 20633346
nitric oxide; S-nitroso-L-cysteine; S-nitrosylation; S-nitrosation; neurotransmitter release; energetic metabolism; brain
5.  Hemoglobin Interactions with αB Crystallin: A Direct Test of Sensitivity to Protein Instability 
PLoS ONE  2012;7(7):e40486.
As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.
PMCID: PMC3399823  PMID: 22815750
6.  Effect of nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine, Spermine and PAPA NONOate on intracellular pH in cardiomyocytes 
Previous studies have suggested that exogenous nitric oxide (NO) and NO-dependent signalling pathways modulate intracellular pH (pHi) in different cell types, but the role of NO in pHi regulation in the heart is poorly understood. Therefore, in this study we investigated the effect of NO donors S-nitroso-N-acetyl-DL-penicillamine, Spermine and PAPA NONOate on pHi in isolated rat ventricular myocytes.The cells were isolated from the hearts of adult Wistar rats, and pHi was monitored using a pH-sensitive fluorescent indicator 5-(and-6)-carboxy SNARF-1 with a confocal microscope. To test the effect of NO donors on sodium-hydrogen exchanger, basal pHi in Na+-free buffer and pHi recovery from intracellular acidosis after an ammonium chloride prepulse were monitored. The role of carbonic anhydrase was tested using acetazolamide. Cl−-OH− and Cl−-HCO3− exchangers were inhibited with 4,4 diisothiocyanatostilbene 2,2' disulfonic acid.All three NO donors acutely decreased pHi. This effect lasted until NO donor was removed. In a Na+-free buffer decrease in basal pHi was increased, while inhibition of carbonic anhydrase and Cl−-OH− and Cl−-HCO3− exchangers did not change the effect of NO donors on pHi. After an ammonium preload, pHi recovery was accelerated in the presence of NO donors.In conclusion, exogenous NO decreased the basal pHi leading to increased activity of sodium-hydrogen exchanger. Carbonic anhydrase and chloride-dependent sarcolemmal HCO3− and OH− transporters are not involved in the NO-induced pHi decrease in isolated rat ventricular myocytes.
PMCID: PMC3430815  PMID: 22703333
Nitric oxide; intracellular pH; cardiac myocytes; sodium hydrogen exchanger; carbonic anhydrase
7.  Differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species: implications for Multiple Sclerosis 
Journal of neurochemistry  2009;109(1):93-104.
Depending on its concentration, nitric oxide (NO) has beneficial or toxic effects. In pathological conditions, NO reacts with superoxide to form peroxynitrite, which nitrates proteins forming nitrotyrosine residues (3NY), leading to loss of protein function, perturbation of signal transduction, and cell death. 3NY immunoreactivity is present in many CNS diseases, particularly Multiple Sclerosis (MS). Here, using the high flux NO donor, spermine NONOate, we report that oligodendrocytes are resistant to NO, while motor neurons are NO sensitive. Motor neuron sensitivity correlates with the NO-dependent formation of 3NY, which is significantly more pronounced in motor neurons as compared to oligodendrocytes, suggesting peroxynitrite as the toxic molecule. The heme-metabolizing enzyme, heme-oxygenase-1 (HO-1), is necessary for oligodendrocyte NO resistance, as demonstrated by loss of resistance after HO1 inhibition. Resistance is reinstated by peroxynitrite scavenging with uric acid further implicating peroxynitrite as responsible for NO sensitivity. Most importantly, differential sensitivity to NO is also present in cultures of primary oligodendrocytes and motor neurons. Finally, motor neurons cocultured with oligodendrocytes, or oligodendrocyte-conditioned media, become resistant to NO toxicity. PRELIMINARY STUDIES SUGGEST OLIGODENDROCYTES RELEASE A SOLUBLE FACTOR THAT PROTECTS MOTOR NEURONS. Our findings challenge the current paradigm that oligodendrocytes are the exclusive target of MS pathology.
PMCID: PMC2756289  PMID: 19226373
motor neurons; oligodendrocytes; nitric oxide; peroxynitrite; nitrotyrosine; hemoxygenase 1
8.  Interactions between adenosine, angiotensin II and nitric oxide on the afferent arteriole influence sensitivity of the tubuloglomerular feedback 
Adenosine, via activation of A1 receptors on the afferent arteriole (AA), mediates the tubuloglomerular feedback (TGF) mechanism. Angiotensin II and nitric oxide (NO) can modulate the sensitivity of the TGF mechanism. However, the interaction among these substances in regulating the TGF resetting phenomenon has been debated. Studies in isolated perfused AA have shown a biphasic response to accumulating doses of adenosine alone. In the nanomolar range adenosine has a weak contractile effect (7%), whereas vasodilatation is observed at high concentrations. However, a synergistic interaction between the contractile response by adenosine and that of angiotensin II has been demonstrated. Adenosine in low concentrations strongly enhances the response to angiotensin II. At the same time, angiotensin II in physiological concentrations increases significantly the contractile response to adenosine. Moreover, addition of a NO donor (spermine NONOate) to increase NO bioavailability abolished the contractile response from combined application of angiotensin II and adenosine. These mutual modulating effects of adenosine and angiotensin II, and the effect of NO on the response of AA can contribute to the resetting of the TGF sensitivity.
PMCID: PMC3714451  PMID: 23882224
adenosine; angiotensin II; tubuloglomerular feedback; afferent arteriole; kidney
9.  Effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide with the estrogen response element. 
Nucleic Acids Research  1997;25(12):2396-2402.
We studied the effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide (ODN1) harboring the estrogen response element (ERE) by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis. Putrescine and spermidine had no marked effect on the CD spectrum of ODN1. In contrast, spermine provoked and stabilized two characteristic changes in the CD spectrum. The first change was indicated by an increase in the intensity of the CD band at 280 nm at 0.5 mM spermine in Tris-HCl buffer containing 50 mM NaCl. This change appears to be related to changes in base tilt and conformational alterations similar to A-DNA. At 1-2 mM spermine, the CD spectrum was characterized by a loss of positive bands at 220 and 270 nm. This change might have contributions from polyamine-induced condensation/aggregation of DNA. Spectral measurements were also conducted in Tris-HCl buffer containing 150 mM NaCl to minimize contributions from condensation and aggregation of ODN1. Under these conditions, CD spectral changes were retained by (ODN1), although the magnitude of the change was diminished. In contrast, a control oligdeoxyribonucleotide (ODN2) having similar base composition did not show any significant change in the CD spectrum in the presence of 150 mM NaCl and 2 mM spermine. The changes in the CD spectrum of ODN1 were highly sensitive to polyamine structure, as evidenced by experiments using spermine analogs with altered number of -CH2- groups separating the amino and imino groups. Electrophoretic mobility shift analysis further showed ODN1 stabilization by spermine and its analogs. These data demonstrate the ability of an ODN containing ERE to undergo conformational transitions in the presence of polyamines and suggest a possible mechanism for polyamine-mediated alterations in the interaction of estrogen receptor with ERE.
PMCID: PMC146762  PMID: 9171091
10.  Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: Role of protein kinase G nitration 
Journal of cellular physiology  2011;226(12):3104-3113.
Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO−) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO− donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24h (with exogenous activation of cGMP) revealed increased ONOO− levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO− and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.
PMCID: PMC3527072  PMID: 21351102
Peroxynitrite; cell signaling; pulmonary hypertension; nitration
11.  Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei 
Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.
PMCID: PMC3517295  PMID: 22521523
antibiotics; antimicrobials; melioidosis; reactive nitrogen species; therapy; [4Fe-4S] clusters
12.  Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in Campylobacter jejuni and Campylobacter coli 
Journal of Bacteriology  2004;186(16):5332-5341.
Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.
PMCID: PMC490904  PMID: 15292134
13.  Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells 
The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing β-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable β-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in β-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes β-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.
PMCID: PMC1752226  PMID: 17205106
apoptosis; nitric oxide; insulin producing cell; TAB1; p38 MAPK
14.  Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619 
The over-production of superoxide (O2⋅−) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI2) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O2⋅− formation, p47phox and Rac1 activation in human vascular smooth muscle cells (hVSMCs) was investigated.
hVSMCs were incubated with 10 nM thromboxane A2 analogue, U46619 for 16 h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1 h and O2⋅− measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac1 was studied using pull-down assays.
NONOate and iloprost inhibited O2⋅− formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac1 and p47phox activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors.
NO and PGI2 block the acute activity of NOX in hVSMCs via the cGMP–PKG axis (for NO) and by the cAMP–PKA axis (for iloprost) through inhibition of Rac1 and p47phox translocation. These findings have implications in the pathophysiology and treatment of CVD.
PMCID: PMC2850987  PMID: 18420399
15.  Nitric Oxide Antagonizes the Acid Tolerance Response that Protects Salmonella against Innate Gastric Defenses 
PLoS ONE  2008;3(3):e1833.
Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.
Methodology/Principal Findings
The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment.
Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.
PMCID: PMC2266805  PMID: 18350168
16.  Nitric Oxide Mediates Tightening of the Endothelial Barrier by Ascorbic Acid 
Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with Nω-nitro-L-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.
PMCID: PMC3022079  PMID: 21156160
paracellular transport; nitric oxide; endothelial permeability; endothelial nitric oxide synthase; guanylate cyclase
17.  Peroxynitrite Increases Protein Phosphatase Activity and Promotes the Interaction of Phospholamban with Protein Phosphatase 2a in the Myocardium 
High levels of peroxynitrite have been shown to decrease cardiomyocyte contraction through a reduction in phospholamban (PLB) phosphorylation. However, previous reports did not examine the direct effect of peroxynitrite on protein phosphatase activity in the myocardium or the role of specific phosphatases. Here we test the effect of the peroxynitrite donor SIN-1 on protein phosphatase activity in whole heart homogenates, as well as the interaction of PLB with protein phosphatase 1 (PP1) and 2a (PP2a). SIN-1 (200 μmol/L) induced a significant increase in protein phosphatase activity, which was alleviated with the specific PP1/PP2a inhibitor okadaic acid. Conversely, lower concentrations of SIN-1 and the nitric oxide donor spermine NONOate (300 μmol/L) were both without effect on phosphatase activity. We next examined the effect of SIN-1 on the interaction of PLB with PP1 and PP2a using co-immunoprecipitation, since okadaic acid inhibited the effects of SIN-1 in our current and previous studies. SIN-1 significantly increased the interaction of PLB with PP2a, but had no effect on the interaction between PLB and PP1. Urate, a peroxynitrite scavenger, inhibited the effects of SIN-1 on phosphatase activity and the interaction of PLB with PP2a, thus implicating peroxynitrite as the causal species. The results of this study provide further insight into the mechanism through which high levels of peroxynitrite serve to decrease PLB phosphorylation and myocardial contraction. Therefore, peroxynitrite signaling could play a key role in the contractile dysfunction manifested in heart failure where peroxynitrite production and protein phosphatase activity are increased and PLB phosphorylation is decreased.
PMCID: PMC2909675  PMID: 20664715
Heart failure; Co-immunoprecipitation; Phosphorylation
18.  Enhancement of thermal killing by polyamines V. The response of EMT6 multicellular tumour spheroids versus monolayer cells. 
British Journal of Cancer  1983;47(1):51-55.
Polyamines, especially spermine, are very effective in enhancing thermal killing of mammalian cells cultured as a monolayer. The response of EMT6 multicellular tumour spheroids to heat in the presence of spermine was studied using cell survival and growth delay as endpoints. Compared to cells in a monolayer, spheroids were found to be highly resistant to combined heat and spermine. In spite of this, considerable enhancement of thermal killing by spermine was observed when the combined treatment was prolonged for a few hours. These results, together with data obtained using labelled spermine, suggest that difficulties in penetration of spermine into the inner cells of the spheroids contribute to the resistance of the latter. A method of circumventing this difficulty is discussed.
PMCID: PMC2011250  PMID: 6821633
19.  Heme Oxygenase-1 Induction Depletes Heme and Attenuates Pulmonary Artery Relaxation and Guanylate Cyclase Activation by Nitric Oxide 
This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-1 (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24 hour organ culture with 0.1 mM cobalt chloride (CoCl2) or 30 μM Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by ~2–4 fold, and lowered heme levels by 40–45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 μM chromium mesoporphyrin during the 24 hour organ culture (but, not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the β1-subunit of sGC. However, the increase in activity seen in the presence of 1 μM spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-1 had decreased levels of superoxide detected by the chemiluminescence of 5 μM lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO.
PMCID: PMC2441442  PMID: 18178725
cGMP; cobalt protoporphyrin; chromium mesoporphyrin; superoxide
20.  L-Ornithine Derived Polyamines in Cystic Fibrosis Airways 
PLoS ONE  2012;7(10):e46618.
Increased arginase activity contributes to airway nitric oxide (NO) deficiency in cystic fibrosis (CF). Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.
PMCID: PMC3465344  PMID: 23071598
Cardiovascular research  2007;74(3):515-525.
C-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarizing factor (EDHF) in the mammalian resistance vasculature, important in the regulation of local blood flow and systemic blood pressure. Additionally, this peptide has been shown to protect against ischaemia-reperfusion injury and inhibits leukocyte and platelet activation. Herein, we use a novel, selective natriuretic peptide receptor-C (NPR-C) antagonist (M372049) to highlight the pivotal contribution of CNP/NPR-C signalling in the EDHF-dependent regulation of vascular tone and investigate the mechanism(s) underlying the release and biological activity of CNP and EDHF.
In vitro pharmacological investigation was conducted in rat (Sprague-Dawley) aorta and mesenteric resistance arteries. Relaxant responses to CNP, atrial natriuretic peptide (ANP), the nitric oxide donor spermine-NONOate (SPER-NO) and the endothelium-dependent vasodilator, acetylcholine (ACh) were examined in the absence and presence of M372049 or inhibitor cocktails shown previously to block endothelium-dependent dilatation in the resistance vasculature. RT-PCR was employed to characterize the expression of NPR subtypes in the vessels studied.
M372049 produced concentration-dependent inhibition of the vasorelaxant activity of CNP in rat isolated mesenteric resistance arteries but not aorta; in contrast, M372049 did not affect relaxations to ANP or SPER-NO in either vessel. M372049 or ouabain alone produced small, significant inhibition of EDHF-dependent relaxations in mesenteric arteries and in combination acted synergistically to abolish such responses. A combination of M372049 with established inhibitors of EDHF-dependent relaxation revealed that multiple, distinct pathways coordinate the bioactivity of EDHF in the resistance vasculature, and that CNP/NPR-C signalling represents a major component.
These data substantiate CNP/NPR-C signalling as a fundamental pathway underlying EDHF-dependent regulation of vascular tone in the rat mesenteric resistance vasculature. An increased understanding of the physiological roles of CNP/NPR-C signalling in the vasculature (now facilitated by the identification of a selective NPR-C antagonist) should aid determination of the (patho)physiological importance of EDHF and might provide the rationale for the design of novel therapeutics.
PMCID: PMC3503309  PMID: 17391657
Blood flow; natriuretic peptide; endothelium-derived hyperpolarizing factor; microcirculation; vasodilation
22.  Aggregation Kinetics of Recombinant Human FVIII (rFVIII) 
Journal of pharmaceutical sciences  2005;94(9):2023-2029.
The physical phenomenon of aggregation can have profound impact on the stability of therapeutic proteins. This study focuses on the aggregation behavior of recombinant human FVIII (rFVIII), a multi-domain protein used as the first line of therapy for hemophilia A, a bleeding disorder caused by the deficiency or dysfunction of factor VIII (FVIII). Thermal denaturation of rFVIII was investigated using circular dichroism (CD) spectroscopy and size exclusion chromatography (SEC). The dependence of unfolding on heating rate indicated that the thermal denaturation of the protein was at least partly under kinetic control. The data was interpreted in terms of a simple two-state kinetic model, N(Native)→kA(Aggregated), where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation. Analysis of the data in terms of the above scheme suggested that under the experimental conditions used in this study, the rate-controlling step in the aggregation of rFVIII may be a unimolecular reaction involving conformational changes.
PMCID: PMC2574007  PMID: 16052549
rFVIII; physical instability; multi-domain; irreversible denaturation; kinetic control
23.  Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum) 
Annals of Botany  2009;104(1):9-17.
Background and Aims
Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum).
NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings.
Key Results
Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N2 flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate.
The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein.
PMCID: PMC2706727  PMID: 19376780
Nitric oxide; nitrate; nitrate reductase; post-translational regulation; tomato; Solanum lycocarpum
24.  Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions 
Nature protocols  2006;1(6):2527-2535.
Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy (ΔH) and entropy (ΔS) of unfolding, the midpoint of the unfolding transition (TM) and the free energy (ΔG) of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength takes approximately eight hours.
PMCID: PMC2752288  PMID: 17406506
25.  Ligand-induced Closure of Inward Rectifier Kir6.2 Channels Traps Spermine in the Pore 
The Journal of General Physiology  2003;122(6):795-805.
Small organic amines block open voltage-gated K+ channels and can be trapped by subsequent closure. Such studies provide strong evidence for voltage gating occurring at the intracellular end of the channel. We engineered the necessary properties (long block times with unblock kinetics comparable to, or slower than, the kinetics of gating) into spermine-blocked, ATP-gated (N160D,L157C) mutant KATP channels, in order to test the possibility of “blocker trapping” in ligand-gated Kir channels. Spermine block of these channels is very strongly voltage dependent, such that, at positive voltages, the off-rate of spermine is very low. A brief pulse to negative voltages rapidly relieves the block, but no such relief is observed in ATP-closed channels. The results are well fit by a simple kinetic model that assumes no spermine exit from closed channels. The results incontrovertibly demonstrate that spermine is trapped in channels that are closed by ATP, and implicate the M2 helix bundle crossing, or somewhere lower, as the probable location of the gate.
PMCID: PMC2229588  PMID: 14638936
spermine; ATP; inward rectifier; gating; K channel

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