Imaging Modalities in Inflammatory Breast Cancer (IBC) Diagnosis: A Computer-Aided Diagnosis System Using Bilateral Mammography Images.

Inflammatory breast cancer (IBC) is an aggressive type of breast cancer. It leads to a significantly shorter survival than other types of breast cancer in the U.S. The American Joint Committee on Cancer (AJCC) defines the diagnosis based on specific criteria. However, the clinical presentation of IBC in North Africa (Egypt, Morocco, and Tunisia) does not agree, in many cases, with the AJCC criteria. Healthcare providers with expertise in IBC diagnosis are limited because of the rare nature of the disease. This paper reviewed current imaging modalities for IBC diagnosis and proposed a computer-aided diagnosis system using bilateral mammograms for early and improved diagnosis. The National Institute of Cancer in Egypt provided the image dataset consisting of IBC and non-IBC cancer cases. Type 1 and Type 2 fuzzy logic classifiers use the IBC markers that the expert team identified and extracted carefully. As this research is a pioneering work in its field, we focused on breast skin thickening, its percentage, the level of nipple retraction, bilateral breast density asymmetry, and the ratio of the breast density of both breasts in bilateral digital mammogram images. Granulomatous mastitis cases are not included in the dataset. The system's performance is evaluated according to the accuracy, recall, precision, F1 score, and area under the curve. The system achieved accuracy in the range of 92.3-100%.

Untargeted Metabolite Profiling of Cerebrospinal Fluid Uncovers Biomarkers for Severity of Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2, Batten Disease).

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.

Bridging medical simulation with computer science and engineering: A growing field of study.

BACKGROUND: Medical simulation has become an essential educational tool in the curricula of healthcare professionals. A literature review revealed a knowledge gap in healthcare simulation education with respect to the technological expertise required to operate highly sophisticated simulation equipment. With this motivation, a case study was designed to determine if implementing on-site technological expertise allows for the facile navigation of high fidelity manikins. Next, a research study was conducted to evaluate engineering students understanding of simulation, and their interest to attend a program in medical simulation. OBJECTIVES: To determine if on-site technological expertise lifts barriers associated with manikin use and to assess levels of understanding and interest among engineering students following exposure to the technology used in healthcare simulation. DESIGN: A prospective, descriptive study with pre-post surveys. SETTINGS: The Nursing Skills and Simulation Center at a New England University campus. PARTICIPANTS: Engineering students attending 6 different engineering programs (Computer Science, Computer Engineering, Mechanical Engineering, Biomedical Engineering, Electrical Engineering and Technology Management) and having different educational levels (undergraduate and graduate). METHODS: Two assessments were applied to engineering students attending a class on technology used in healthcare simulation. A pre-test measured the understanding and interest of students in the engineering/computer science courses before attending a simulation class. A post-test assessment measured their improvement in understanding and interest to learn more about simulation technologies. RESULTS: Statistical analysis and comparisons of pre-and post-test assessments show a 23% gain in understanding of this field following exposure to the healthcare simulation class. Furthermore, post test results show greater than 67% of those surveyed have an interest in attending a program in healthcare simulation. CONCLUSIONS: The results indicate the collaboration of nursing and engineering has lifted known barriers to simulation education, and reveal engineering students have an interest in the field of medical simulation.

Repair Mechanisms in Oxidant-Driven Chronic Inflammatory Disease.

The interplay that governs chronic diseases through pathways specifically associated with chronic inflammation remains undefined. Many metabolic events have been identified during the injury and repair process. Nonetheless, the cellular events that control the pathogenesis of inflammation-induced disease have not been fully characterized. We and others reason that chronic inflammatory diseases associated with a cascade of complex network mediators, such as nitric oxide, arachidonic acid metabolites, cytokines, and reactive oxygen species, play a significant role in the governance of alterations in homeostasis, oxidative stress, and thromboatherosclerosis. In this context, we discuss lipid mediators associated with the maintenance of health, including the specialized proresolving mediators that help drive cellular repair. Emphasis is placed on the pathophysiology of chronic metabolic insults involving both the airways and the cardiovascular system during oxidant-driven inflammatory disease. In this review, we highlight new pathways of inquiry that show promise for the identification of those metabolic targets that can improve therapy for chronic inflammation.

Pulmonary Abnormalities in Young, Light-Use Waterpipe (Hookah) Smokers.

RATIONALE: Waterpipes, also called hookahs, are currently used by millions of people worldwide. Despite the increasing use of waterpipe smoking, there is limited data on the health effects of waterpipe smoking and there are no federal regulations regarding its use. OBJECTIVES: To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers. METHODS: We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles. MEASUREMENTS AND MAIN RESULTS: Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles. CONCLUSIONS: Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking.

Smoking-Associated Disordering of the Airway Basal Stem/Progenitor Cell Metabotype.

The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography-mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium.

Serum Metabolite Biomarkers Discriminate Healthy Smokers from COPD Smokers.

COPD (chronic obstructive pulmonary disease) is defined by a fixed expiratory airflow obstruction associated with disordered airways and alveolar destruction. COPD is caused by cigarette smoking and is the third greatest cause of mortality in the US. Forced expiratory volume in 1 second (FEV1) is the only validated clinical marker of COPD, but it correlates poorly with clinical features and is not sensitive enough to predict the early onset of disease. Using LC/MS global untargeted metabolite profiling of serum samples from a well-defined cohort of healthy smokers (n = 37), COPD smokers (n = 41) and non-smokers (n = 37), we sought to discover serum metabolic markers with known and/or unknown molecular identities that are associated with early-onset COPD. A total of 1,181 distinct molecular ions were detected in 95% of sera from all study subjects and 23 were found to be differentially-expressed in COPD-smokers vs. healthy-smokers. These 23 putative biomarkers were differentially-correlated with lung function parameters and used to generate a COPD prediction model possessing 87.8% sensitivity and 86.5% specificity. In an independent validation set, this model correctly predicted COPD in 8/10 individuals. These serum biomarkers included myoinositol, glycerophopshoinositol, fumarate, cysteinesulfonic acid, a modified version of fibrinogen peptide B (mFBP), and three doubly-charged peptides with undefined sequence that significantly and positively correlate with mFBP levels. Together, elevated levels of serum mFBP and additional disease-associated biomarkers point to a role for chronic inflammation, thrombosis, and oxidative stress in remodeling of the COPD airways. Serum metabolite biomarkers offer a promising and accessible window for recognition of early-stage COPD.

Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase.

Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.

Inducible nitric oxide synthase provides protection against injury-induced thrombosis in female mice.

Nitric oxide (NO) is an important vasoactive molecule produced by three NO synthase (NOS) enzymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). While eNOS contributes to blood vessel dilation that protects against the development of hypertension, iNOS has been primarily implicated as a disease-promoting isoform during atherogenesis. Despite this, iNOS may play a physiological role via the modulation of cyclooxygenase and thromboregulatory eicosanoid production. Herein, we examined the role of iNOS in a murine model of thrombosis. Blood flow was measured in carotid arteries of male and female wild-type (WT) and iNOS-deficient mice following ferric chloride-induced thrombosis. Female WT mice were more resistant to thrombotic occlusion than male counterparts but became more susceptible upon iNOS deletion. In contrast, male mice (with and without iNOS deletion) were equally susceptible to thrombosis. Deletion of iNOS was not associated with a change in the balance of thromboxane A(2) (TxA(2)) or antithrombotic prostacyclin (PGI(2)). Compared with male counterparts, female WT mice exhibited increased urinary nitrite and nitrate levels and enhanced ex vivo induction of iNOS in hearts and aortas. Our findings suggest that iNOS-derived NO in female WT mice may attenuate the effects of vascular injury. Thus, although iNOS is detrimental during atherogenesis, physiological iNOS levels may contribute to providing protection against thrombotic occlusion, a phenomenon that may be enhanced in female mice.

The scavenger receptor CD36 contributes to the neurotoxicity of bone marrow-derived monocytes through peroxynitrite production.

CD36, a class B scavenger receptor present in microglia, endothelium and leukocytes, plays a key role in ischemic brain injury by promoting the expression of inflammatory genes and production of reactive oxygen species (ROS). However, it is not known whether ischemic brain damage is mediated by CD36 activation in resident brain cells, i.e., microglia, or by blood-borne cells that infiltrate the brain. To address this question, we studied oxygen-glucose deprivation (OGD) in hippocampal slice cultures, a model of ischemic injury that does not involve cells extrinsic to the brain. We found that CD36 gene knockout does not afford protection of hippocampal slices to OGD-induced cytotoxicity. In contrast, immunoactivated bone marrow-derived monocytes-macrophages (BMM) from wild type (WT) mice trigger hippocampal damage when incubated with brain slices via a mechanism that is prevented in CD36-/- BMM. The neurotoxic activity of CD36+/+ BMM was attributed to reactive oxygen species (ROS) since it was concomitant with increased ROS production and could be prevented by treatment with a selective ROS scavenger, MnTBAP, or a peroxynitrite decomposition catalyst, FeTPPS. Importantly, ROS production and accumulation 3-nitrotyrosine in hippocampal proteins (a hallmark of peroxynitrite production) was significantly dampened in immunoactivated CD36-/- BMM, whereas production of NO-derived metabolites (nitrite and nitrate) was unaltered. We conclude that CD36 signaling may not contribute to injury induced by OGD in the brain itself but is involved in the neurotoxicity mediated by activated BMM. These findings are consistent with the hypothesis that CD36 in infiltrating inflammatory cells drives peroxynitrite-mediated ischemic brain damage. Accordingly, targeting CD36 in the vascular compartment may protect against neurotoxicity in the ischemic brain.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli.

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

Physical evidence for substrate binding in preventing cyclooxygenase inactivation under nitrative stress.

Prostaglandin biosynthesis is catalyzed by two spatially and functionally distinct active sites in cyclooxygenase (COX) enzymes. Despite the crucial role of COXs in biology, molecular details regarding the function and regulation of these enzymes are incompletely defined. Reactive nitrogen species, formed during oxidative stress, produce modifications that alter COX functionalities and prostaglandin biosynthesis. We previously established that COX-1 undergoes selective nitration on Tyr385 via a mechanism that requires the presence of bound heme cofactor. As this is a critical residue for COX-1 catalysis, nitration at this site results in enzyme inactivation. We now show that occupancy of the COX-1 active site with substrate protects against Tyr385 nitration and redirects nitration to alternative Tyr residues on COX-1, preserving catalytic activity. This study reveals a novel role for the substrate in protecting COX-1 from inactivation by nitration in pathophysiological settings.

Silent Partner in Blood Vessel Homeostasis? Pervasive Role of Nitric Oxide in Vascular Disease.

The endothelium generates powerful mediators that regulate blood flow, temper inflammation and maintain a homeostatic environment to prevent both the initiation and progression of vascular disease. Nitric oxide (NO) is arguably the single most influential molecule in terms of dictating blood vessel homeostasis. In addition to direct effects associated with altered NO production (e.g. vasoconstriction, excessive inflammation, endothelial dysfunction), NO is a critical modulator of vaso-relevant pathways including cyclooxygenase (COX)-derived prostaglandin production and angiotensin II generation by the renin-angiotensin system. Furthermore, NO may influence the selectivity of COX-2 inhibitors and ultimately contribute to controversies associated with the use of these drugs. Consistent with a central role for NO in vascular disease, disruptions in the production and bioavailability of NO have been linked to hypertension, diabetes, hypercholesterolemia, obesity, aging, and smoking. The ability of the vessel wall to control disease-associated oxidative stress may be the most critical determinant in maintaining homeostatic levels of NO and subsequently the prospect of stroke, myocardial infarction and other CV abnormalities. To this end, investigation of mechanisms that alter the balance of protective mediators, including pathways that are indirectly modified by NO, is critical to the development of effective therapy in the treatment of CV disease.

Protein 3-nitrotyrosine in complex biological samples: quantification by high-pressure liquid chromatography/electrochemical detection and emergence of proteomic approaches for unbiased identification of modification sites.

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues.

Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase.

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH(4)). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH(4) becomes oxidized in organs of apolipoprotein E-deficient (ApoE(-/-)) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH(4) were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE(-/-) mice or ApoE(-/-) mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE(-/-) mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH(4) oxidation in heart, lung, and kidney of atherogenic ApoE(-/-) mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE(-/-) mice and that these species differentially contribute to protein 3-NT accumulation and BH(4) oxidation in a tissue-selective manner. Since BH(4) oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.

Reprint of "oxidative alterations of cyclooxygenase during atherogenesis" [Prostag. Oth. Lipid. M. 80 (2006) 1-14].

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NOx) species modulate cyclooxygenase (COX; also known as prostaglandin H2 synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO-) has multiple effects on COX activity. ONOO- can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO- results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO- can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE-/- mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE-/- mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO- persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO- and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NOx species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NOx species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.

Oxidative alterations of cyclooxygenase during atherogenesis.

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NO(x)) species modulate cyclooxygenase (COX; also known as prostaglandin H(2) synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO(-)) has multiple effects on COX activity. ONOO(-) can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO(-) results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO(-) can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE(-/-) mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE(-/-) mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO(-) persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO(-) and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NO(x) species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NO(x) species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite.

The mechanism by which the inflammatory enzyme prostaglandin H(2) synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO(-)) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO(-) exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO(-), heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO(-). Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO(-) only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO(-) and the demise of PGHS-1.