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Two more to go. If we hurry we may be able to save all of them. Follow me! I can guard and operate the elevators while you search for the workers. Take this key and use it to release the first worker. He will have another key to give you when you free him. I will operate the elevators. After you free the first three I'll take you down to find the other two.

I'll stay here and keep the area secure. Find the trapped men and bring them back to me. Now you need to find the other two workers. When you're done I'll take you to the next floor down by the elevator. There is only one more worker trapped on this floor. Find him and I will activate this elevator to the floor below. I'll start the elevator now. Take this key, it's for the locked doors down below.

Please, be very careful. Remember, there are two good men down there. You saved us all! Please, go see the foreman. I'm sure he has a fine reward for your efforts. Let's go. Thank you for finding me. Hello, I'll follow you. Lead the way out! Thank you! I feel much better now.

I'm safe. Thank you. That was too close! I'll stop here. Please take me back to the elevator. Please, help me back to the elevator. I used this key to lock myself in here. Take it. You need it to release another trapped worker. Go and find the other workers. Don't worry about me, I'm safe here. We couldn't have made it without your help.

I Am Wolf (The Children of Nox, #1) by Joann Buchanan

All the workers have been found. We are in your debt. You saved our lives.


Hecubah was right, men are evil! Pay for your crimes against Mother Nature! We have a horrible problem! Horrific overgrown insects and airborne demons from the underworld have overrun the mines. Mine workers have died in defense of Nox's vital interests! Please, we beg you! Clear the beasts from the mines, rescue my miners and I shall reward you handsomely. This key will unlock the door to the first trapped mine worker. He will help you find the others. I also have a beast scroll which should be quite useful. If you need a bow or quiver, you can use the ones found in my quarters.

The entrance to the mines is straight down this path. My deepest thanks. While you were in the mines, saving my men, the Captain came by, looking for you. He's waiting for you at the Crossroads -- east of here. Before you go, please take this gold as reward. Just follow the main path. You would be wise to listen to his advice. One more to go. Great Job!. Con04a Con04a: CaptainGreet The entrance to the tombs is in the building at the end of the path.

See if Hecubah's been here. You'll know. And careful. Many of the crypts have traps against grave robbers. We must know if reports of Hecubah's acts are true. Go into the crypts to see if Hecubah has already been here. Con04a: NecroDies Arrrggh! You may have stopped me, but you'll never leave this crypt alive. Con04a: NecroGloats Now your flesh will rot and your corpse will serve Hecubah. Con04a: NecroTaunts Cling to your life of flesh while you can! You'll soon be free of its shackles.

Con04a: NecroThreatens It will be your honor to serve Hecubah, human, but first you need to die. Con04a: NecroThreatens2 To the dust you'll return, only to be raised again in the ranks of our undead army. Con04a: NecroWarnsHec Hecubah, beware, human eyes are upon us. Con05 Con Bartender01 Have a drink, stranger. The ogres took the women, but at least they left the cider. Con Bartender02 Ogres burned the village.

Have a drink? Con Bartender03 This used to be such a nice neighborhood -- 'till the ogres moved in. Con Bartender04 What's that you say? You're going to fight the ogres? Well good luck to ya. It's about time somebody stood up to them. Con Bartender05 What can I get ya, stranger? Con BartenderTalk01 Have a drink, stranger. Ogres burned the village. This used to be such a nice neighborhood -- 'till the ogres moved in. What's that you say? What can I get ya, stranger? Con OgreKingTalk01 You are very bold for such a little man.

Con OgreKingTalk02 Too bad you must die now. Con OgreKingTalk05 I will crush your bones! Con OgreKingTalk06 Yummie! Ha ha ha Con OgreKingTalk07 Din' din'. Come and get it! Con OgreTalk01 Intruder! Seal off the King's Hut! Con OgreTalk02 What 'dat noise? Con OgreTalk03 Mmmm Smells like lunch!

Con OgreTalk04 Shut 'dem gates! Con OgreTalk05 Now let's get 'im! Con OgreTalk07 You have been quite the trouble maker Now you must be Punished. Con OgreTalk08 This is for your own good. Con Townsman10Talk01 Oh no I've lost everything. Con Townsman11Talk01 This is a terrible tragedy. Con Townsman11Talk02 My home Con Townsman9Talk01 The ogres have destroyed everything!

Maybe we shouldn't have built so close to the ogre village. The ale's fresher'n a monkey's butt! What can I get fer you? You got a problem, buddy? Get on the other side of the bar like everyone else! If yer not gonna buy anything, get the hell outta 'ere! What are you doing? Watch it, buddy. Watch it, I'm trying to keep an eye on that dog! Those stupid Ogres turned my wife into a dog!

Have you seen my staff? Is there something you would like to see? Care for some exotic wares from faraway mystical lands? Would you like to purchase something? How are you doing? Please, watch your step. Be careful! There are flames everywhere! It's all fun and games until one of us gets burned! Oh, I'm so worried about Lewis! Normally customers stand on that side of the counter. Are you planning on buying anything or are you just looking? Excuse me. Watch where you're going! Watch it, buddy! Oh, I didn't see you there! Sorry about that. But no time to rest on laurels.

You must now recover the Amulet of Teleportation, which the Ogres took from Horvath when they raided the unlucky Hamlet of Brin!

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With the Ogres on the move and with this as their prize, Hecubah is most certainly behind it all. Horvath is waiting in the village. Now, off with you! He is waiting to talk to you. Be off with you! Hecubah is on the move and we need to stop her! Wha'sh your name? Wha'sh that you shay? I don't feel so good You have completed mission 5! Please, help me, a horrific fate has befallen my wife!

Not long ago, a raving demoness led the Ogres through here. She cast an evil spell, turning my poor wife into a wolf! I could reverse this curse if I only had my magic staff, but I left it in my house northwest of here. I'm afraid if I leave to go retrieve it, my wife will run off! Would you help me by fetching my staff for me, kind sir? I will offer a worthy reward for its timely return. Of course I don't need your help to remove the curse on my wife! Some lucky adventurer will get a fine reward, though. It's you again If you have so much time on your hands, why won't you get my magical staff back from that Ogre?

Many thanks to you, Wanderer. Your noble deeds will be long remembered in these parts! I can never hope to repay you fully for your service, but please take this as a token of our appreciation! I need my magical staff! Please hurry, sir! My wife needs help immediately! Get my staff back soon and I shall give you a nice reward!

The curse has been broken! I'm free!!!! We will never forget your selfless deeds! Good luck on your journey, kind stranger! Lead me to safety! The fires are getting hotter! I will follow you, just get me out of here! It's not easy being green. Rescue me! Get your exotic wares here!! What can I interest you in? I am thankful you are here at last, for we are in dire need of your help. The Ogre forces overran Brin a short time ago and robbed me of the Amulet of Teleportation!

Please go to the Ogre village in the northeast and retrieve the precious artifact as soon as possible! Thanks to your efforts, the Amulet has been returned safe and sound. Congratulations on your success, Apprentice! Many thanks for your aid in rescuing the Amulet! With its return, we will finally be able to thwart that foul demoness' plans once and for all! Here, these bottles of Mana and the Infravision spell will come in handy Please go swiftly! For given the chance, Hecubah will use the Amulet for the darkest purposes, indeed!

Find the precious Amulet of Teleportation and return it safely! That cursed Ogre village is somewhere northeast of here. Pant, Pant If I don't get it back, he'll be destroyed. He's supposed to arrive soon! Please, help me, kind sir! I think the Ogres who took it went to the docks. Could you Hello there again! You're welcome in my house anytime! Thank you so much for helping me! Please accept this as a token of my appreciation! My father will be home soon! I hope you find the cloak soon. If you can get my Father's cloak back, I have something I can give you as a reward!

Do you have my Father's cloak yet? I think the Ogres ran off toward the docks! You will probably find the cloak with some Ogres out near the docks. Mission Failed. Mission Incomplete. Lewis is I'm inconsolable. The Ogres have set my house on fire and my poor frog, Lewis, is trapped by the flames in the other room! Rescue him and I'll forever be indebted to you! Hello there! Many thanks for saving my precious frog! Hello again, Wanderer! How can I ever repay you for your bravery?! Loads and loads of money? Well, if that's what you require, please take this as a sign of my appreciation.

Just hurry and rescue Lewis! Please, save my frog, Lewis, from the fire! The fires are raging! I can barely stand the smoke in here! Please, save Lewis! We carry the finest wares in all of Nox! Maybe not the finest exactly But damn good nonetheless! Hey if you see any Ogres 'round here, lead 'em to us, ok? My sword arm needs some action. My arm's crampin' up.

I wish there were some monsters to fight. A gypsy -- name of Loproc -- just passed through here recently. If you hurry up to Brin, you might catch him before he moves on. A friendly warning if you are traveling north; we have been stationed here to guard against Ogre attacks. A large band of these foul creatures just laid waste to the poor Village of Brin north of here Watch out for the Ogre War Lord, he throws shurikens!

Everything was fine till those wretched Ogres showed up Those Ogre brutes are huge! Don't you have more important things to be doing? The gypsy Loproc is in town again! Did you hear what happened to poor Thavius' wife? Those damn Ogres made off with almost everything! Get 'em! Here, these bottles of Mana and the Infravision spell will come in handy -- sooner than you think. Please travel swiftly, for given the chance, Hecubah could use the Amulet for the darkest purposes, indeed!

Come here ya little runt! Get in my belly! Time to die! Catch that human! Needs food Where are all the others? Where is everybody? Run for it! He's stronger than he looks! Watch out! He has mighty magic! Didn't even scratch me! Keep it comin', little man! That didn't even leave a mark! At last, a challenge! You think your puny weapons can hurt me? I will squash you like a bug! I will break you! Dungeon Entrance: Your Dungeon taxes at work. Future dungeon expansion planned. Dungeon Entrance: Consult your local utility company before digging.

Dungeon Entrance: Wipe you feet before entering. Dungeon Entrance: If you were imprisoned here, you'd be home by now. Fight on for the glory of Horrendous! Con SaveDunMir05 We must press on and avenge those that have fallen this day! Thanks for your help, brave Conjurer! May your flame burn bright on this dark night, Fire Knight! I shall take the field wherever Hecubah pleases, it does not matter where. But then I shall throw down the challenge of single combat which no one may refuse.

Once I alone face her, she shall understand the true meaning of Warlord. The sorceress, Hecubah, has attacked us with an army of Undead. All hands to the fortress! We must safeguard Horrendous. Hecubah must NOT get his halberd. Go to the fortress and report! Hecubah has the power to raise the dead. We must stop her before she grows stronger. We must defend the fortress! Defense of the fortress is imperative! Con06a: NecroAttackJack You were warned. Now the price of this folly will be your gravestone! Con06a: NecroDies A curse on your blood forever! Con06a: TownspeopleSpeak2 Out of my way!

You're supposed to defend us, you coward! We need the fortress to hold strong! What will this do to our economy? All is lost! How do we stop someone that death cannot? Con LeavingGalava1 The Ogres are marching to destroy us. The smart people have already left Galava. If you're smart you'll leave Galava, too. Let them take it, I say. Con MiscMage01 Lead on, brave Conjurer.

Our combined Magics will repel these vile creatures. Its power must not fall into evil hands! Con MiscMage03 Hecubah must have discovered the secrets of teleportation. One minute the tower was clear and the next it was flooded with ogres and demons! They want the Heart of Nox. Con SaveDunMir07 We have done it! The Fire Knights have never burned so brightly as on this glorious day!

Now we must gather our flame around Horrendous and bring the fight to Hecubah herself! The Heart of Nox is located in the topmost floor of the Tower of Illusion. Hecubah's ogres have already overrun the lower portions of the Tower. Guildmaster Horvath has not been heard from since the invasion began. You'll be on your own. Once you retrieve the Heart of Nox, get back here as quickly as you can. Good luck! Over here! You in need of a new bow? Some arrows, maybe? I think I may have a few Conjurer spells on hand. I've got the finest assortment of arms in the South! Galava isn't exactly a vacation spot right now, what with Ogres running rampant in the Tower.

I'm just about ready to pick up and move out. Everything's on sale, if you're interested. You're going to need magical help, lad. I'm willing to lower my prices if you think you can reclaim our Tower for us. The finest Inn of the Southern Lands! You'd best get back home. No doubt Hecubah will be attacking Ix soo, too! Chaotic times we live in, isn't it? The Wizards locked the gates to their Tower and all kinds of strange sounds have been coming out of there. Something's horribly wrong.

I hear Hecubah's armies are on the march. Time for me to think about setting up shop elsewhere. These grounds are safe haven. Even Ogres wouldn't violate the sanctity of St. Allistor's church! I can't believe the wizards lost control of their Tower. Bad tidings indeed!

Better stock up while you can! I hope you can toss a few Ogre bodies my way. I wouldn't mind burying a few of them myself! The Ogres have taken over the Tower! I have heard the Heart of Nox has a complex and dangerous security system. Watch out for Ember demons. Their fireballs are lethal! Please help us! The Heart of Nox is beyond your reach! You do not want to face me in combat! I command thee! Obliterate him! You can't stop me Horvath! I have the power to destroy you now -- worthless old fossil! And next time, you will not have Horvath's lackeys to protect you!

There is another, more powerful than me, who now stands in your way. The Heart of Nox is here. Go, with the blessings of the Mages' Guild. With Horvath gone, Hecubah will now have greater freedom to create deadly havoc! You must quickly retrieve the Heart of Nox for the staff. Good luck, Conjurer. Con08 Con AldwynProd Go now!

Our time grows short. Con AldwynProd2 I am proud to see a Conjurer carry the weapon which can bring an end to Hecubah's madness. You must go to the temple and speak to the priests. Here is the key to unlock the gate which blocks passage to the temple. Con08a: CaptainProd This is as close as I can bring you.

The Temple of Ix lies north of the village. You must make your way quickly, but you may need the help of Aldwyn to get there. Retrieve the Weirdling Beast and meet me back here in this clearing. Move smartly lad! Con08a: CemeterySign Cemetery closed until further notice. Curse Hecubah! Con08a: GuardGreet Welcome home, young Conjurer. Aldwyn is looking for you.

Well done, young Conjurer! Con08a: Mystic2 Perhaps you could use a scroll or potion? Con08a: PriestGreet We have been expecting your arrival. The sacred Order of Oblivion are the caretakers and devout guardians of the Weirdling. Once you reach the Weirdling the beast will cling to the staff, since the Heart of Nox contains the richest source of energy it requires -- much more than the meager offerings in the pools.

Con08a: PriestProd The Weirdling thrives on pure energy, which it receives from the Xon Pools in the remote depths of our subterranean temple. Con08a: PriestProd2 With the energy provided by the staff, the Weirdling Beast can channel the very forces of life itself. It is a weapon without peer and will even channel a bit of your adversaries' life into your own. Con08a: PriestProd3 It is time to resume your quest, lad, Hecubah grows more powerful with each passing minute. I'll require gold to cover the cost of your spellbook.

Con08b: InversionBoyTalk02 Very good. Now when my assistant casts a spell you invert it back.

Use the mana obelisk to recharge. Be careful, we don't unlock the gates until you invert them all. Con08b: InversionBoyTalk03 Excellent. Try a few more. Con08b: InversionBoyTalk04 I'm sorry but you need gold. Con08b: InversionBoyTalk04a Hmmm, too bad. Basic training isn't for everyone. Con08b: InversionBoyTalk05 Very good. Con08b: InversionSign Inversion lessons - Today only! Con09 Con DryadThreat1 Hecubah said your kind would come here seeking to destroy her, but I'll put an end to your little plan! Con DryadThreat2 Step no closer, human!

I knew you'd come to defile this place -- just like everything else you touch. Con DryadThreat3 You are no match for my magic. The creatures at my command are more than you can handle. Con09a: MordwynHome Make yourself at home, I'll be right back. Con09a: MordwynHome2 Ah, here they are My brother brought them here for safekeeping, in the event Ix was overrun. They may prove most useful against the dangers you'll surely face to the North. Con09a: NecroThreat1 Your quest is futile, weakling! You'll not contaminate our land with your cursed living flesh!

Con09a: NecroThreat2 Pathetic Slug In this mire you will remain! Con09a: NecroThreat3 No living thing dare sets foot within the halls of Hecubah's hallowed temple. Con09a: NecroThreat4 Choose death now and join the front lines of the proud Fallen That vile pink flesh of yours will soon return to the earth. You have completed mission 10! So now, the Ogres will feed on your flesh tonight!

You are on my land now. Meddle any further into my business and you'll pay with your life. Now you must pay the ultimate price for your insolence. My minions will teach you a harsh lesson -- you'll sadly have scant time to remember since your pathetic death is imminent! This time you sorely over- estimate yourself!

Unfortunately you'll not live long enough to comprehend the depths of your hearty foolishness! My minions and I will destroy you with ease! This time you've crossed the line -- puny mortal! That puny stick is not going to help you here Con10B. You're in my domain now! Come to me, my Dark Minions of Pain! By themselves they are an annoyance, but in a swarm they can rip you to bits in seconds. A bolt of pure energy emanates from these ocular guardians. They can only be found in the frozen wastelands of Nox.

They hurl pure fire and, when near death, will explode in a furious blaze. Their Lord harnesses the force of nature in his staff. They are faster and more powerful than their natural counterpart. Awkward and slow, their kick packs a wallop. Don't hang around after one dies, their stench is noxious. They are much more dangerous than the common zombie. They are much more dangerous than they appear. They have little love for humans. Skilled conjurers can train them to hunt. They are the sad foot soldier of the Undead.

Guess I'll have to wait a bit to talk. Do you want to wait or exit now? Please close all other applications and restart Nox. Do you wish to overwrite? You may use the slider to adjust the rate at which data is sent to other players. GeneralDialog: TownsmanHello Hello there! All players must enter to warp. GeneralPrint: AdvanceToStage2 To Warp, one or more active players must have previously completed the destination stage or higher.

Share next time! You have been punished for your lack of cooperation! GeneralPrint: BluePotion Use blue potions to refresh mana. See your Journal for details. GeneralPrint: EliminationCantJoin Players have already been eliminated, you must wait until the next map to join. GeneralPrint: GainedItem An item has been added to your inventory!

GeneralPrint: GainedKey A key has been added to your inventory! GeneralPrint: CharmHint Doors will unlock after you have charmed a spider. GeneralPrint: IllegalItem Illegal items were detected on your character and have been removed. Players now share Silver Keys. Key sharing does not apply to Treasure Chests, or Gold Locks.

GeneralPrint: LockBroken1 The lock has been broken! GeneralPrint: LockBroken2 You have broken the lock! Would you like to pay a penalty and continue, or quit the game? Quit now? GeneralPrint: PressKey Press any key to continue. Your score has been reset and a character penalty has been assessed. Would you like to continue or quit the game? You will not be able to join the adventure until someone leaves the game. GeneralPrint: RedPotion Use red potions to restore health. You have just used up one of your Ankhs.

GeneralPrint: SecretFound You have uncovered a secret area! GeneralPrint: ShopHints Fighting hordes of monsters alone may be overwhelming. Try to stay close to your friends and fight as a team. Your combined efforts will be much more powerful. Don't hog all your loot! If you share the wealth with your friends, they will be able to buy better equipment, which will make the whole party stronger.

Sell me any items you don't need, or can't use. You can then use the gold to buy better equipment for yourself. Buy all the potions you can carry! Adventures can be long, and help may be hard to find. Summoning Obelisks must be destroyed or they will spawn hordes of evil monsters and overwhelm you!

I Am Wolf - The Children of Nox Series Book 1

Try to destroy Summoning Obelisks right away, then fight any monsters that are left behind. If you don't, the Obelisks may create more monsters before you defeat the first ones! Match your attacks with your foes. Some monsters are immune to certain attacks, such as fire. If you are clever, fight from afar. Warriors shouldn't forget their shurikens and chakram.

There are secret areas hidden everywhere! Some are hidden behind breakable sections of walls, others across dangerous terrain, and others require a clever mind If you find yourself overwhelmed by evil monsters, run away to fight another day. Also make sure you never run into unfamiliar areas - there may be even more dangerous foes that way! Search for magical Ankhs! If you find one, you will gain an extra life. Once your last Ankh is used, your adventure is over.

It is wise to protect your friends so they can protect you in return. Use your keys wisely. Gold keys are very rare, and are used to enter special areas. You will find both riches and danger behind gold doors, however. Use your automap to keep track of your friends and find mana. It is also useful for locating areas you have yet to visit. Sometimes I sell different items to each player.

Shop with your friends - you might find more than you expected. Beware of Hecubah and her Necromancer minions! Eat food and use red potions to keep yourself healthy and strong at all times. You never know what may be lurking around the next corner It inhibits not only all of the NOX isoforms, but also nitric oxide synthase , xanthine oxidase , mitochondrial complex I , and cytochrome P reductase Studies on the effect of the phagocyte NADPH oxidase suggested that apocynin 1 must be metabolized by peroxidases to generate the inhibitory compound and 2 acts by inhibiting the translocation of cytoplasmic subunits According to statement by Lafeber et al.

While these initial studies on apocynin appear solid, a low-affinity prodrug is not an ideal drug candidate, nor is it a powerful tool for research. Nevertheless, apocynin is now used indiscriminately as a bona fide NOX inhibitor. Apocynin at extremely high concentrations has been used as a NOX4 inhibitor e.

Apocynin is also widely used in cell types where there is no evidence for the presence of a peroxidase. A final complicating factor in the use of apocynin comes from recent studies which suggest that apocynin may actually stimulate ROS production in nonphagocytic cells , Neopterin is a pteridine that is generated by macrophages as a catabolic product of GTP, and serum levels are elevated during some cancers and during human immunodeficiency virus HIV. However, neopterin is not specific inhibition of xanthine oxidase, Ref.

The peptide inhibitor gp91ds-tat was designed specifically to inhibit NOX2 by mimicking a sequence of NOX2 that is thought to be important for the interaction with p47 phox. Also, as the region targeted by the peptide is homologous in other NOX isoforms, the peptide may lack specificity. In summary, there are currently no potent and specific NOX inhibitors, and studies that base their conclusions solely on the use of the above-described compounds should be taken with some caution.

In this section, we discuss our present knowledge of polymorphisms for each of the NOX family members. The mutations in NOX2 and its subunits that lead to CGD are rare disease-causing mutations and not polymorphisms and are therefore not included. This analysis is based on current data base entries, not all of which have been confirmed, thus the precise numbers should be taken with caution. Nevertheless, an interesting picture is beginning to emerge. When normalized by gene size, the differences are much smaller, ranging from 2. When looking at coding nonsynonymous SNPs, the differences become more marked: the number of coding nonsynonymous SNPs per 1, amino acids is Thus there are differences in the degree of amino acid conservation among different NOX isoforms and subunits.

The degree of conservation of most NOX isoforms and subunits is high, suggesting an essential role of these enzymes. The relatively low conservation of NOX5 together with its loss in rodents suggest that mammalian organisms depend less on this isoform. The relatively low conservation of p22 phox is puzzling, given the fact that this subunit interacts with four NOX isoforms. Polymorphisms in p22 phox affect NOX2 activity, and it is tempting to speculate that p22 phox variability is a cause of biological variations in ROS generation in the human population.

It has been suggested that the number of these dinucleotide repeats regulates NOX2 expression and activity Yet present databank entries suggest only one coding nonsynonymous SNP within the human gene, namely, an IleMet polymorphism. For mammals, NOX5 is not an essential gene, as the gene is lacking in rodents see above. In line with this observation, NOX5 in humans seems to be more permissive to genetic variations than other NOX family members.

Currently eight entries with coding nonsynonymous SNPs are found in the database. The latter polymorphism is caused by a C to T substitution and is often also referred to as the CT polymorphism Most studies suggest a decreased ROS generation in phagocytes , or vascular cells from individuals with the His72Tyr polymorphism, yet one study in granulocytes describes the opposite Studies on the effect of the His72Tyr polymorphism on cardiovascular disease yielded contradictory results.

One study also reports equal risk for cardiovascular risk factors, but increased salt sensitivity and decreased levels of nitric oxide metabolites in His72Tyr carriers The AG substitution has been linked to a protective effect in cardiovascular disease and an improved response to exercise through an enhanced reduction of oxidative stress markers AG is also linked to a protective effect against drug-induced agranulocytosis The -GA gene is associated with higher promoter activity The promoter variant -GA is associated with hypertension , and an increased level of oxidative stress , Note that, at least in the caucasian population, the His72Tyr polymorphism is not in linkage disequilibrium with the -GA polymorphism ; thus it is difficult to unequivocally attribute an observed phenotype to one of the two polymorphisms.

In summary, research on the clinical impact of p22 phox polymorphisms is at an early stage. A synopsis of results obtained with the most studied polymorphism, His72Tyr, has not yielded a clear trend, suggesting that it is not a major risk factor, at least for cardiovascular diseases. The results concerning the AG and the -GA polymorphism are less contradictory, but the number of studies is small. There are currently four SNP entries for p67 phox in the databank. Three other polymorphisms, not included in databank entries, Vallle, ProSer, and HisGln, were identified by systematic sequencing of p67 phox from healthy individuals In a reconstituted Cos7 cell system, these variants show a functional activity similar to wild-type p67 phox Thus, presently, there is no indication that p67 phox polymorphisms are of functional relevance.

There are four database entries for p40 phox SNPs. Details regarding the proposed physiological and pathophysiological function of NOX NADPH oxidases will be given in the sections on the respective organs and systems. The orientation of NOX enzymes is such that the NADPH binding NH 2 terminus is in the cytoplasm, and superoxide generation occurs either in the extracellular or an intraorganellar space. B : H 2 O 2 is a well-established signaling molecule, which readily permeates biological membranes.

Biological effects of NOX-derived H 2 O 2 include the following: 1 oxidation of low p K a cysteine residues, which leads to inactivation of protein tyrosine phosphatases, disulfide bridge formation e. Host defense might also be an important role of other NOX family members. However, while a direct ROS-dependent killing was initially thought to completely explain the host defense function of NOX enzymes, it has become clear that the situation is more complicated and that several mechanisms cooperate to achieve successful oxygen-dependent killing.

It is also important to remember that phagocytes have a number of oxygen-independent killing mechanisms, in particular, the release of microbicidal proteins from the granules reviewed in Ref. Indeed, studies with neutrophils from CGD patients demonstrate that NOX2 is not required for the killing of many types of bacteria As a general rule, however, catalase-positive organisms e. When the phagocyte respiratory burst was discovered, a direct killing of microorganisms by ROS was proposed.

However, the type of ROS that actually kill bacteria is still debated. Although superoxide is the species produced by NOX enzymes, it is not clear whether superoxide itself is directly involved in killing of microorganisms. Thus under some physiologically relevant conditions, such as the low pH in the phagosome and the nonpolar environment close to cell membranes, superoxide itself could potentially be a direct player in killing. Once generated, superoxide dismutates into hydrogen peroxide, either spontaneously, particularly at low pH, or facilitated by superoxide dismutase.

Therefore, bacterial killing by hydrogen peroxide derived from superoxide has been suggested as a mechanism for NOX activity. A role in bacterial killing has also been suggested for DUOX2-derived hydrogen peroxide in combination with lactoperoxidase in the digestive tract , , The combined effect of hydrogen peroxide and myeloperoxidase has been extensively studied, and it is clear that this system is powerful in killing bacteria and in inactivating bacterial pathogenicity factors.

Killing by this system involves different types of peroxidation reactions, which have been discussed previously , Within the phagosome, HOCl appears to be produced in sufficient concentrations to kill bacteria However, while myeloperoxidase-deficient individuals are at an increased risk of infection , most are healthy, unlike the much more severe immunosuppression found in CGD patients deficient in NOX2.

Thus, while myeloperoxidase undoubtedly provides an important amplification of NOX2-dependent killing mechanisms, it is not the only, and perhaps not even the major, mechanism. Superoxide avidly reacts with nitric oxide to form the highly reactive intermediate peroxynitrite, which in turn can be metabolized to several other highly RNS. The relative importance of RNS in killing by phagocytes is unknown, but it is possibly more important in rodents than in humans Nonenzymatic conversion of superoxide and hydrogen peroxide can also lead to the formation of highly reactive free radicals.

Catalyzed by iron, hydrogen peroxide can produce the hydroxyl radical in a reaction known as the Fenton reaction While this reaction occurs readily in vitro, the extent to which it occurs in vivo is less clear. Indeed, evidence suggests that at least in neutrophils, the lack of an iron catalyst limits the amount of hydroxyl radical formed by Fenton-like reactions Yet, the hydroxyl radical may still be formed in these cells through an iron-independent reaction between superoxide and hypochlorous acid catalyzed by myeloperoxidase Hydrogen peroxide can also react with superoxide to produce the hydroxyl radical and singlet oxygen , or with hypochlorous acid to produce singlet oxygen and harmful chloramines Indeed, bactericidal activity of phagocytes from CGD patients is reconstituted by the addition of a glucose oxidase system capable of generating peroxide and hypochlorous acid Rather than killing of microorganisms directly, NOX enzymes may also act through inactivation of bacterial virulence factors.

There is ample evidence for inactivation of virulence factors by the hydrogen peroxide myeloperoxidase system , However, more recent studies suggest that such an inactivation may also occur through redox-sensitive elements within a given virulence factor. The weight of evidence supports the idea that ROS generation by NOX2 is an important mechanism for killing of microbes and inactivation of microbial virulence factors see above. However, recently an alternative concept has been proposed.

This concept suggests that a direct effect of ROS is not crucial and that NOX2-dependent changes of phagosomal pH and ion concentrations are responsible for killing. The NOX2 elevation of the phagosomal pH is probably due to the fact that superoxide is a weak base and that protons are consumed when superoxide dismutates into hydrogen peroxide , NOX2-dependent phagosome alkalinization might allow for optimal function of neutral proteases in neutrophils In dendritic cells, prolonged NOX2-dependent phagosomal alkalinization plays an important role in the cross-presentation of antigens As an electron transporter, activation of NOX enzymes leads to a charge build-up that requires compensation , In summary, most likely the contribution of NOX2 to microbial killing lies in both direct ROS effects and indirect effects through modulation of pH and ion homeostasis.

Electron transport by NOX enzymes leads to a charge build up, which requires compensation. Thus there is no doubt that there is a close relationship between NOX enzymes and proton channels; however, there are two different molecular interpretations: 1 NOX enzymes possess a proton channel domain, suggested to reside within the histidine-rich third transmembrane domain 55 , 57 , , , , , and 2 NOX enzymes are closely associated and interact with physically distinct proton channels , Candidates for such a distinct proton channels are the recently described voltage sensor-domain proteins , Since ROS are usually associated with inflammation, an anti-inflammatory activity of NOX enzymes seems counterintuitive.

However, over the recent years there has been an impressive number of publications pointing in this direction.

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It may seem surprising that most data on an anti-inflammatory activity of NOX enzymes come from studies using mice deficient in the phagocyte NADPH oxidase NOX2, p47 phox , which typically is considered a prototype proinflammatory enzyme. But one should also remember that the name chronic granulomatous disease see sect.

Indeed, chronic granulomas in CGD patients are thought to be sterile complications , and in CGD mice, a sterile hyperinflammation can be caused by injection of sterilized Aspergillus fumigatus extracts This hyperinflammatory response might be due to the following: 1 a decreased capacity to degrade phagocytosed material in NOX2-deficient cells leading to the accumulation of debris , ; 2 lack of ROS-dependent signaling in NOX2-deficient phagocytes, e. Hyperinflammation in phagocyte NADPH oxidase-deficient mice is not limited to hyperinflammation in response to inactivated A.

It is also observed in mouse models of Helicobacter gastritis 90 , , arthritis , , demyelinating disease , and sunburn In experimental lung influenza infection, NOX2-deficient mice show heightened inflammatory infiltrates and accelerated viral clearance Even more strikingly, genetic determination of the disease locus in arthritis-prone mice and rats identifies p47 phox mutations as the underlying defect. These observations in animal experiments might relate to clinical observations in CGD patients, where in addition to infections, patients with CGD suffer from a variety of inflammatory conditions , In some instances, these inflammatory disorders are the first clinical manifestation of CGD.

There is also a high incidence of lupus erythematosus in family members of CGD patients In summary, there is mounting evidence that NOX enzymes have a role in limiting the inflammatory response. This anti-inflammatory activity of NOX enzmyes represents an interesting and surprising function. The underlying molecular mechanisms await further studies. The best understood, and possibly most important, pathway by which ROS achieve regulation of cell function occurs through redox-sensitive cysteine residues.

This has been most convincingly demonstrated for protein tyrosine phosphatases PTP. PTPs control the phosphorylation state of numerous signal-transducing proteins and are therefore involved in the regulation of cell proliferation, differentiation, survival, metabolism, and motility The catalytic region of PTPs includes cysteines 60 , , which are susceptible to oxidative inactivation Thus ROS decrease phosphatase activity that enhances protein tyrosine phosphorylation and thereby influences signal transduction Consistent with this biochemical mechanism, NOX-derived ROS have been shown to regulate protein tyrosine phosphorylation in several different cell types , , , Treatment of cells with hydrogen peroxide leads to phosphorylation and activation of p38 mitogen-activated protein MAP kinase There is abundant evidence for activation of elements of the MAP kinases system by NADPH oxidases , , , , , , , see also examples given in sect.

The precise redox-sensitive step s involved in kinase activation in response to NOX-dependent ROS production is presently unknown. ROS can regulate intracellular and plasma membrane ion channels. Such a regulation of ion channels may occur either directly or through ROS-sensitive signaling systems. NOX isoforms have been increasingly implicated in this function, but it should be kept in mind that other sources of ROS may also be important Unfortunately, no data from NOX-deficient animals exist, and the evidence therefore remains circumstantial.

NOX enzymes may regulate the activity of such channels through two major mechanisms: ROS-dependent posttranslational modifications e. S -glutathiolation is a posttranslational modification of protein cysteines mediated by the interaction of peroxynitrite derived from nitric oxide and superoxide and glutathione, which ultimately leads to the formation of a reversible disulfide bond between the protein and glutathiolation A stronger oxidative stress leads to an irreversible oxidation of thiols and thereby to enzyme inhibition There is abundant evidence for the regulation of gene expression by ROS.

Most studies investigating the mechanisms of mRNA upregulation in response to ROS have concluded that transcriptional upregulation is the underlying cause. This upregulation can occur through redox-sensitive second messenger systems e. A large number of studies describe cell death in response to NOX activation Table 3. ROS can trigger apoptosis either indirectly, through damage to DNA, lipids, and proteins or more directly by ROS-mediated activation of signaling molecules. MAP kinase activation occurs in many instances through ROS-dependent inhibition of tyrosine phosphatase At higher ROS concentrations, hydrogen peroxide can inhibit caspases and thereby lead to a switch from apoptosis to necrosis , TABLE 3.

NOX enzymes as pro- or antiapoptotic. The role of NOX enzymes can be regarded as either pro- or antiapoptotic and may depend on the stimulus, the model, or the method of study. It has also been suggested that superoxide is a natural inhibitor of Fas-mediated cell death Thus NOX activation is most commonly associated with cell death; however, under certain circumstances it may be antiapoptotic. Possible reasons for such apparently contradictory functions include 1 the magnitude and duration of the ROS signal, 2 the subcellular localization of the respective NOX isoform, 3 the set of redox-sensitive signaling targets e.

Similar to what is described above for cell death and survival, there are arguments that NOX-derived ROS may lead to either cellular senescence or to enhanced cell growth. ROS are thought to be a key mechanism in the aging process 73 , , and there is abundant evidence for an acceleration of cellular senescence through oxidative stress In the light of these observations, it is not surprising that several studies report NOX induction of cellular senescence and cell cycle arrest , Yet, despite the well-established role of ROS in cellular senescence, there is also evidence that under many circumstances ROS can accelerate cell growth A first hint came from a study showing that many rapidly growing tumor cells release large amounts of ROS Subsequently, many studies have documented a role of ROS as second messengers in cell proliferation 9 , , , , , , , , , , ; in most of these studies there was at least circumstantial evidence that the source of mitogenic ROS was an NADPH oxidase.

Thus, when new NOX enzymes were detected, the question of whether they are involved in the regulation of cell proliferation resurfaced. It was suggested that hydrogen peroxide mediates the cell growth and transformation caused by the Nox1 Later, however, the authors of these studies detected the presence of V12 RAS in their cell lines, suggested that the observed transformation was probably due to RAS, and cautioned against the use of these cells Indeed, NOX expression in other fibroblasts failed to produce transformation Nevertheless, there is now a significant number of studies suggesting an involvement of NOX enzymes in cell proliferation.

In vitro studies based on either antisense or siRNA suppression suggest a role of NOX4 and NOX1 in smooth muscle cell proliferation , , , a role of NOX5 in proliferation of esophageal adenocarcinoma cells , and a role for p22 phox in proliferation of endothelial cells Note, however, that angiotensin II-induced aortic smooth muscle proliferation was conserved in NOX1-deficient mice Thus a critical review of the literature concerning the relationship between NOX enzymes and cell proliferation suggests that 1 there is abundant evidence for a regulation of cell proliferation in vitro through reactive oxygen species, 2 in vitro knock-down experiments argue in favor of NOX enzymes being involved in the regulation of cell proliferation, and 3 there is so far no convincing data from knockout mice suggesting that NOX enzymes play a crucial role for cell proliferation in vivo.

Probably all of our cells are capable of sensing the ambient oxygen concentration and responding to hypoxia. However, some organs are specialized in oxygen sensing, particularly the kidney cortex, the carotid bodies, and the pulmonary neuroepithelial bodies. At least two cellular events allow cells to detect hypoxia Fig. In the case of HIF, under normoxic conditions, HIF prolyl hydroxylases mediate HIF hydroxylation at specific prolines and thereby promote its rapid degradation 8 , Under hypoxic conditions, this process is inhibited leading to stabilization of the HIF protein.

While the hydroxylase is undoubtedly a directly oxygen-dependent enzyme, there is good evidence that increased ROS generation under hypoxic conditions can also contribute to HIF stabilization. The ROS effects may be mediated through oxidation of divalent iron, which is an obligatory cofactor for the hydroxylase.

Under normoxic conditions, CO is generated by hemoxygenase through an oxygen- and P reductase-dependent breakdown of heme. Under normoxic conditions, HIF is hydroxylated, which leads to its rapid degradation. Traditionally, NOX enzymes were thought to be involved in oxygen sensing through a decreased ROS generation in hypoxic tissues. However, many recent results led to a revised model where hypoxia increases ROS generation. Traditionally NOX enzymes were thought to be involved in oxygen sensing through a decreased ROS generation in hypoxic tissues.

A reduction in ROS generation during hypoxia is also observed in isolated perfused lungs 31 , Thus, although it is hard to extrapolate from these measurements to the in vivo situation, it is conceivable that under hypoxic conditions, ROS generation by NOX enzymes drops. A revised model where hypoxia increases ROS generation is depicted in Figure 6. Such a hypoxia-induced ROS generation, while counterintuitive, has now been amply documented , , , , There are solid arguments for both suggestions.

In favor of a role of mitochondria, suppression of the mitochondrial cytochrome- c oxidase suppresses hypoxia-induced ROS generation in various cell lines , , In favor of a role of NOX enzymes, studies using primary mouse carotid body chemoreceptor cells demonstrate that moderate hypoxia leads to increased ROS generation that is absent in cells derived from p47 phox -deficient mice There are no mechanistic experiments demonstrating a role of NOX4 in oxygen sensing by the kidney, but such a role is conceivable based on the NOX4 localization in the kidney cortex , Carotid bodies are sensory organs composed of a small cluster of cells located near the bifurcation of the carotid artery.

Carotid bodies detect changes in arterial oxygen saturation and respond to hypoxia by inducing tachycardia and increased ventilation. The carotid body is composed primarily of two cell types: the glomus cells, which act as the primary oxygen-sensing cells, and the sustentacular cells, which surround the innervated glomus cells Molecules and mechanisms implied in oxygen sensing in the carotid body include hemoxygenase , nitric oxide synthetase , the mitochondrial respiratory chain 45 , a direct ion channel modulation by oxygen , , and NOX enzymes.

Early theories suggested a role for NOX2, which can be detected in carotid bodies 7 , , ; however, the presence of NOX2 in carotid bodies probably reflects the presence of macrophages Several studies suggest normal oxygen sensing in NOX2-deficient mice 32 , , , and there are no reports of any oxygen-sensing deficits in patients with CGD. However, while NOX2 deficiency does not alter oxygen sensing, there are two studies suggesting an altered oxygen-sensing in p47 phox -deficient mice , In this system, there is relatively strong evidence for an involvement of NOX2 in oxygen sensing: in NOX2-deficient mice, there is a decreased response to hypoxia in newborn animals and in cells from neuroepithelial bodies This NOX2 dependence of oxygen sensing appears to be specific for the pulmonary neuroepithelial bodies, as oxygen sensing was not impaired in pulmonary artery smooth muscle from NOX2-deficient mice Peroxidation reactions are important in physiology.

One of the best-documented roles for NOX enzymes is the iodination of thyroid hormones, a reaction catalyzed by the thyroid peroxidase using DUOX-derived hydrogen peroxide , , Another example is the H 2 O 2 and peroxidase-dependent cross-linking of dityrosine residues in the extracellular matrix, which has been shown to be DUOX-dependent in cutaneous tissue of Caenorhabditis elegans Dityrosine cross-linking is also important for the hardening of the fertilization envelope in sea urchin eggs, where it is mediated by the DUOX homolog Udx1 Whether NOX-dependent dityrosine cross-linking is also important in mammalian tissues is unknown.

Disulfide bond formation is determined by the redox potential.

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Thus NOX-dependent ROS generation might possibly have a broad influence on protein disulfide bridges in the entire cell or in restricted cellular compartments. Such a role of NOX enzymes has so far received little attention. However, the possible localization of NOX4 in the endoplasmic reticulum , , , the mediation of ER stress through NOX4 , and the association between NOX enzymes and protein disulfide isomerase point towards such a role of NOX enzymes.

Most functions of NOX family members are linked to the reduction of molecular oxygen to superoxide. However, at least in yeast, the FRE homologs within the NOX family reduce metal ions, particularly trivalent iron , Similar to yeast, mammals can absorb divalent but not trivalent iron. Thus a functional ferric reductase activity located within the small intestine has been known and studied for a long time.

The fact that NOX1 is localized in the intestine led to initial speculations that it might be the long sought-after mammalian ferric reductase. However, NOX1 localizes mainly to the colon 55 , , while ferric reductase activity localizes mainly to the small intestine, thus making this possibility rather unlikely. Also, a duodenal cytochrome b , Dcytb, which bears no structural resemblance to NOX enzymes, has been suggested to function as mammalian ferric reductase although this notion has been recently challenged, Ref.

Iron reduction and absorption is not only relevant in the small intestine. Iron starvation of phagocytosed microorganisms is thought to be a host defense mechanism. Removal of iron from the phagosome is thought to occur in a similar manner to iron absorption in the colon, in two steps: 1 reduction of trivalent iron through an oxidoreductase and 2 translocation of divalent iron across the phagosome membrane by a divalent metal transport Nramp1, Ref. It has been suggested that NOX2-derived superoxide could indeed act to reduce iron ; however, presently available data suggest that iron uptake in myeloid cells is NOX2 independent However, while most studies report a stimulatory role of NOX enzymes on matrix metalloproteinase expression, recent in vivo data from NOX2-deficient mice appear to suggest that the absence of NOX2 might also lead to elevated matrix metalloproteinase levels , , Various pathological conditions associated with neovascularization are associated with ROS generation , ROS may directly activate hypoxia-inducible factor Fig.

Angiogenesis-inducing ROS were found to be generated by NOX2 in endothelial cells , and in a model of hindlimb ischemia , but by NOX1 in tumor models of angiogenesis NOX-derived ROS have also been implicated in angiogenesis during differentiation of embryonic stem cells Indeed, superoxide readily reacts with nitric oxide to produce peroxynitrite, thereby decreasing nitric oxide levels , However, it appears that other mechanisms, including uncoupling of the nitric oxide synthase and inhibition of dimethylarginine dimethylaminohydrolase, may also contribute to the antagonistic effect of ROS on the nitric oxide pathway Nitric oxide is an important mediator in a wide range of physiological and pathophysiological processes and is the subject of numerous articles recent reviews include Refs.

The biological impact of nitric oxide modulation by NOX-derived superoxide is widespread and complex. A few examples are outlined here. Nitric oxide and superoxide have opposing effects on vascular tone, and they react with one another negating their individual effects NADPH-dependent ROS generation contributes to the development of nitrate tolerance by depleting nitric oxide and by promoting the uncoupling of nitric oxide synthase LPS stimulation of microglia leads to generation of superoxide by NOX2 and nitric oxide by inducible nitric oxide synthase; these react to form peroxynitrite and lead to cell death in neighboring oligodendrocytes Bradykinin reduces myocardial oxygen consumption through nitric oxide.

NOX2-derived superoxide antagonizes this nitric oxide effect Nitric oxide inhibits T-cell proliferation, and this inhibition has been shown to be reversed by NOX-derived superoxide production A very recent study also suggests a reverse cross-talk through downregulation of NOX1 by nitric oxide In summary, NOX enzymes exert a broad range of actions beyond the direct killing of microorganisms. Most NOX activities are mediated through ROS; however, in some circumstances the electrical driving forces generated by these enzymes may mediate physiological functions in its own right , One of the key activities of NOX-derived ROS is posttranslational modification of proteins in particular phosphatases, transcription factors, and other signaling molecules.

As opposed to earlier concepts, ROS interaction with proteins does not invariably lead to irreversible oxidative damage. Other important aspects of the activity of NOX-derived ROS include the interaction with other macromolecules lipids, carbohydrates, nucleic acids or with small molecules in particular nitric oxide , and also the superoxide-driven alkalinization. Many of the effects described here influence one another. Thus, although there are still many unresolved issues, the molecular basis of physiological NOX functions is now becoming increasingly clear.

For the purpose of this review, we selected organ systems that currently appear most pertinent. The sections are organized as follows: 1 ROS generation in the specific organ or tissue, 2 expression of NOX isoforms, 3 physiological role of NOX, and 4 implication in disease states. ROS generation in adipocytes occurs in response to insulin , , , but has also been described to occur spontaneously and to be enhanced in obese animals Data from 3T3-L1 cells, a fibroblast cell line that can be differentiated into an adipocyte phenotype, are less clear. Interestingly, most studies measuring ROS generation by adipocytes primary cells and cell lines detect release of hydrogen peroxide rather than superoxide, which potentially argues in favor of NOX4 see section on NOX4.

ROS are suggested to enhance adipocyte differentiation In differentiated adipocytes, release of ROS through NOX in response to insulin stimulation activates the distal insulin signaling cascade, including mobilization of glucose transporters to the surface of adipocytes However, prolonged exposure to ROS can decrease glucose transporter expression and interfere with glucose uptake In obese patients, protein-tyrosine phosphatase activity was increased, and insulin-stimulated glucose uptake was decreased Thus ROS production in adipose tissue appears to be a physiologically relevant cellular signaling mechanism in the insulin response, exerting a protective antihyperglycemic action through enhanced adipocyte differentiation and glucose uptake by differentiated adipocytes.

However, if there is a sustained surplus of glucose in the metabolic balance of the organism, NOX activity will contribute to the development of obesity. Probably the earliest descriptions of a respiratory burst came from studies on fertilization , Since then, the generation of ROS by sperm has been demonstrated in a large number of species tested 17 , 49 , , However, some studies suggested that spermatozoa themselves do not possess NADPH oxidase activity 34 , , and that sperm ROS generation might be explained by leukocyte contamination within sperm preparations 22 , , or by mitochondrial ROS generation First, in situ hybridization showed NOX5 mRNA only in very early stages of spermatogenesis, in particular pachytene spermatocytes; the NOX5 protein might be expressed in mature spermatozoa, but this has not been demonstrated.

Second, although NOX5 is found in many mammalian species, it is not found in rodents, while rodent spermatozoa have also been reported to generate ROS. This apparent discrepancy might be explained by the possible expression of NOX2 and its subunits in mouse spermatozoa NOX-derived ROS might be important for maturation of spermatocyte maturation or the function of mature spermatocytes.

In analogy with apoptosis in other cell types in particular neurons and hepatocytes , NOX enzymes might be involved in the mechanisms of apoptosis. Another interesting hypothesis is a potential role for NOX enzymes in cell proliferation and differentiation during spermatogenesis; however, this possibility has not been studied. More data are available on the function of ROS in regulation of activation and function of mature spermatocytes 18 , It has been suggested that NOX-derived ROS regulated acrosome formation and capacitation, the final steps in sperm maturation 20 , 49 , , , , , Finally, NOX enzymes are involved in the respiratory burst that occurs during fertilization , ; in the case of sea urchin eggs, the burst is mediated through the DUOX homolog Udx1.

This respiratory burst is thought to prevent the entry of supernumerary sperms through the stabilization of the fertilization envelope. The biochemical mechanism of this stabilization involves dityrosine cross-linking of proteins within the fertilization envelope. Two important points should, however, be considered in this context. While mammalian strategies to block polyspermy are often similar to those applied by nonmammals , the ROS-dependent stabilization of the fertilization envelope through dityrosine cross-link has to our knowledge not been reported in mammals. Excessive ROS generation might also be implicated in sperm pathology.

Male infertility has been linked to excessive ROS generation In male-factor infertility, oxidative stress is thought to affect the fluidity of the sperm plasma membrane, and ROS-induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts In line with the janus-faced function of ROS in spermatocyte physiology and pathophysiology, there are reports that antioxidants may prevent oxidative damage to sperm but may be detrimental to sperm development at high levels NOX5 was described in prostate cancer cell lines and in prostatic adenocarcinomas The physiological function of ROS generation in prostate cells is little understood, but may be related to cell growth The possible role of ROS in prostate cancer development has received particular attention, because the consumption of the tomato-derived antioxidant lycopene is associated with a decreased prostate cancer risk ROS may lead to cell cycle arrest and apoptosis in vitro, a mechanism that can be limiting for in vitro fertilization However, in vivo, a role for NOX-derived ROS in meiotic maturation of oocytes has been suggested, as maturation was prevented by apocynin and diphenyleneiodonium ROS generation by the endometrial epithelium was observed already more than 20 years ago, and an NADPH oxidase was suggested to be involved ROS levels appear to be increased in the late phase of the menstrual cycle NOX2 expression might be due, at least in part, to the presence of placental macrophages, the so-called Hofbauer cells The physiological role of ROS generation in the placental trophoblast has been suggested to include host defense and degradation of noxious substances or signaling and oxygen sensing Evidence continues to accumulate that oxidative stress contributes to the pathogenesis of preeclampsia pregnancy-induced hypertension Increased ROS generation by neutrophils has been discussed; however, results are contradictory , Microparticles are small membranous vesicles that are released from various cell types.

These microparticles have been suggested to be involved in mechanisms of preeclampsia. Microparticles from women with preeclampsia, but not from controls, led to endothelial dysfunction through ROS-dependent mechanisms , In a different line of thought, it has been suggested that agonistic antibodies against the angiotensin II receptor AT1 in preeclamptic patients stimulates NADPH oxidases in vascular smooth muscle and in the trophoblast In summary, NOX enzymes are expressed in the uterus and the placenta, but little is known about their physiological function in this context.

There is, however, increasing evidence that NOX enzymes are involved in the pathogenesis of preeclampsia. The involvement of NOX enzymes in cardiovascular physiology and pathophysiology has attracted enormous attention, with over articles published on this topic. Several specialized reviews have been published recently 80 , , , , Note also that statins, widely used drugs to treat cardiovascular diseases, have an indirect NOX inhibitory action through inhibition of Rac isoprenylation There is evidence for the involvement of NOX enzymes in cardiac pathophysiology, and it has been suggested that the beneficial effects of statins in cardiac disease may, at least in part, be attributable to NOX inhibition , A protective effect of statins in a stroke model has also been attributed to NOX inhibition ROS generation in the vascular system has been suspected for a long time.

Initially, the source of ROS was thought to be mitochondria and thus an unavoidable side effect of aerobic respiration. Subsequent data, however, suggested an active enzymatic ROS generation, with NOX2, at that time the only known NOX family member, and xanthine oxidase as the main suspects These measurements also quickly established that there were clear differences between the properties of the phagocyte NADPH oxidase and the enzymatic activity observed in vascular preparations, which led to the widespread use of the term vascular NADPH oxidase.

However, there is in fact no vascular specific NOX isoform, but rather a complex expression of different NOX isoforms in different cells and regions of the vascular system Fig. NOX enzymes in the vascular wall. The scheme depicts NOX enzymes in the vascular wall and the text to the right their putative functions. All three layers of the vascular wall [intima i. The effects of NOX in the vascular system might therefore be at least in part due to depletion of the vasorelaxant NO.

In vascular smooth muscle, the message for NOX4 is also abundantly expressed , The protein expression and activity of NOX4 in cerebral arteries appears to be much higher than in systemic arteries While both NOX1 and NOX2 have also been described in vascular smooth muscle, there appears to be a distinct anatomical distribution: the NOX1 message is mainly expressed in large conduit vessels , while NOX2 mRNA is more strongly expressed in resistance vessels , Based on studies in vascular smooth muscle, it has been suggested that NADPH oxidase activity largely depends on NOX4 expression under resting conditions ROS-dependent increase in blood pressure is thought to be a key function of NOX in the vasculature , Strong data in favor of a role of NOX-derived ROS as a hypertensive signaling element come from studies demonstrating a decreased systolic blood pressure response to angiotensin II and to BMP4 in p47 phox -deficient mice.

A decrease in basal blood pressure and blood pressure response to angiotensin II , is also found in NOX1-deficient mice. Studies on blood pressure in NOX2-deficient mice are less conclusive: angiotensin II-induced hypertension is moderately decreased or not changed Evidence for a role of p47 phox in hypertensive signaling comes also from studies in patients with Williams-Beuren syndrome, where p47 phox hemizygosity decreases risk of hypertension Thus, taken together, these studies suggest a predominant role for NOX1, possibly functioning together with p47 phox as an organizer subunit, in ROS-dependent blood pressure elevations.

In the vasculature, NOX-derived ROS are thought to be involved in nitric oxide inactivation , , , growth and cell division , , kinase activation , , activation of matrix metalloproteinases , activation of transcription factors and gene expression , extracellular matrix regulation , endothelial cell proliferation and migration 3 , and neointimal formation The role of NOX family enzymes in vascular pathophysiology has received wide attention.

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The NOX-derived ROS have been implicated in a variety of vascular diseases, including hypertension , , , , , , aortic media hypertrophy , , , atherosclerosis 41 , , , , , , and vascular diabetic complications NOX-derived ROS produced in response to angiotensin II have been implicated in the vascular complications that are associated with insulin resistance Recently, work in NOX2-deficient mice suggests that NOX2-derived ROS are involved in the pathological interaction between blood cells and vessel walls that occurs in sickle cell disease ROS generation in cardiac tissues is low under basal conditions However, it increases in response to activation by various stimuli , , , , Several cell types within the heart contribute to this ROS generation, including cardiomyocytes, endothelial cells, and inflammatory cells , , , , The source of ROS produced by cardiomyocytes was initially suggested to be mitochondria ; however, there is increasing evidence for expression of NOX enzymes in the heart see Table 4.

TABLE 4. Expression of NOX enzymes. Reference numbers are given in parentheses. Globally, NOX function in cardiomyocytes can be divided in a developmental function , , , and a function in adult cardiomyocytes. During development, NOX4 is the predominant isoform and drives cardiac differentiation through activation of p38 MAP kinase In the adult heart, NOX2 is the predominant isoform, and it is involved in the regulation of redox-sensitive signaling cascades, such as modulation of kinases and phosphatases , , , gene expression, protease activation , and superoxide-dependent nitric oxide inactivation The relative importance of NOX enzymes in tissue damage during myocardial infarction remains controversial.

In mouse models of myocardial infarction, there is no decrease in infarct size in NOX2- and p47 phox -deficient animals , Ischemic preconditioning is an interesting concept that might have clinical applications in surgery. The basic observation is the following: preexposure of the heart to a mild stress [e.

Both ischemic and angiotensin II-dependent preconditioning depend at least partially on NOX2: angiotensin II-dependent preconditioning is attenuated by NOX inhibitors , while ischemic preconditioning is completely abolished in NOX2-deficient mice There is also evidence for the involvement of NOX enzymes in cardiac hypertrophy, fibrosis, and progression towards heart failure 79 , , , , , The role of various NOX isoforms in cardiac hypertrophy depends on the stimulus.

In contrast, pressure overload-induced myocardial hypertrophy does not require NOX2, but might possibly involve NOX4 , NOX2-dependent ROS generation is increased in atrial myocytes from patients with a history of atrial fibrillation ROS are generally thought to play an important role in the pathophysiology of septic shock and organ failure ROS derived from NOX2 in neutrophils are thought to be involved in the destruction of hepatocytes and in intestinal tissue damage A possible role for NOX in shock-associated organ failure is suggested by studies where the amount of tissue injury is reduced by NOX inhibitors such as DPI 2 , or apocynin 2 , 82 , Also, platelet-derived exosomes from septic patients lead to apoptosis of endothelial cells in an ex vivo assay Only limited data from knockout mice are available.

In contrast, there is no survival advantage in NOX2-deficient mice after endotoxic shock In summary, it is likely that NOX enzymes are important in shock-related pathologies; however, more in vivo data with different NOX-deficient mice will be needed. The presence of ROS in the central and peripheral nervous systems has received considerable attention over the past several decades. Additionally, the nervous system is particularly sensitive to oxidative stress because of enrichment of polyunsaturated fatty acids in many of the membranes.

NOX4 expression in the brain has been detected by RT-PCR, immunohistochemistry, and in situ hybridization, where it localizes in neurons The principal cellular constituents of the CNS are three cell types of neural descent, neurons, astrocytes, and oligodendrocytes, as well a one cell type of myeloid descent, namely, microglia. In accordance with microglia being a professional phagocyte, a key function of ROS in microglia is thought to be its participation in the host defense and the removal of debris from the CNS. Oligodendrocytes are responsible for myelination in the CNS.

No ROS generation or expression of NOX enzymes in oligodendrocytes has been reported to our knowledge, although these cells are responsive to ROS generated in neighboring cells Astrocytes are glial cells with a complex function within the CNS: they play an important role in providing substrates and regulatory molecules for neurons and also participate in the inflammatory response. NOX-derived ROS are involved in astrocyte intracellular signaling ; they might also be involved in the regulation of cell survival; however, data in that respect are contradictory , Increased NOX2 expression is found in reactive astrocytes hinting towards the possibility that astrocyte NOX2 contributes to oxidative damage in neuroinflammation 6.

In neurons, the expression of ROS-generating NADPH oxidases was considered unlikely for a long time because of their high suseptibility to oxidative damage. However, this paradigm has changed. Globally, two functions of NADPH oxidases in neurons have been proposed: alteration of cell fate and modulation of neuronal activity.

Induction of neuronal apoptosis in response to serum deprivation or by brain-derived neurotrophic factor is mediated by NOX2 , However, NOX enzymes may also modulate neuronal activity. Angiotensin II-stimulated ROS generation , is thought to mediate neuronal chronotropic actions There is also increasing evidence for a role of ROS in cognitive functions, and mice overexpressing superoxide dismutase SOD have impaired memory , More specifically, NOX enzymes, particularly NOX2, might be involved in long-term potentiation and learning , Most importantly, learning and memory are impaired in NOX2- and p47 phox -deficient mice , as well as in CGD patients Note, however, that the degree of learning and memory impairment is mild, suggesting either that NOX2 plays only a modulatory role or that various NOX isoforms have a redundant function.

For example, ROS produced in hippocampal neurons during long-term potentiation diffuse into neighboring oligodendrocytes where they stimulate kinases 40 ; the resulting enhanced phosphorylation of myelin basic protein is thought to enhance myelinization Stroke size was markedly reduced in NOX2-deficient mice , while increased NOX2 expression in diabetic rats was associated with an aggravated ischemic brain injury In a gerbil model of global cerebral ischemia-reperfusion injury, apocynin strongly diminishes damage to the hippocampus There is also increasing evidence for a role of microglial NOX2 in inflammatory neurodegeneration, including Alzheimer's disease and Parkinson's disease , In the case of Alzheimer's disease Fig.

APP fragments released from neurons activate NOX2 in neighboring microglia cells through a Vav-dependent mechanism Several studies suggest similar mechanisms in Parkinson's disease 91 , , ; however, the microglia-activating ligands are less well defined. B : oligodendrocyte death is an early event in the progression of demyelinating diseases. In the case of periventricular leukomalacia, microglia activation, e.

More specifically, lipopolysaccharide LPS leads to activation of microglial inducible NO synthase and microglial NOX2, which generate peroxynitrate through the reaction of nitric oxide with superoxide. Peroxynitrate is a highly reactive oxygen species that is able to kill oligodendrocytes. Similar mechanisms might be involved in other demyelinating diseases such as multiple sclerosis. ROS have also been implicated in the progression of the demyelinating disease Fig. Periventricular leukomalacia is a focal necrosis of white matter in the brain that underlies most cases of cerebral palsy.

In periventricular leukomalacia, the combination of NOX2-derived superoxide and inducible nitric oxide synthase-derived nitric oxide leads to the formation of peroxynitrate and thereby to the killing of oligodendrites The data on the role of the phagocyte NADPH oxidase system in autoimmune encephalomyelitis yielded complex results: depending on the length of the antigen that was injected, p47 phox -deficient or p47 phox mutant mice developed either a less severe or a more severe disease , Also, genetic intercross experiments in susceptible and resistant rats identify a low ROS generating variant of p47 phox as an aggravating factor for autoimmune encephalitis Thus the role of NOX autoimmune encephalitis lies in the boundary between the situation in neurodegenerative disease, where ROS are disease-causing factors and autoimmune disease where ROS appear protective.

There is also increasing evidence for their role in dysfunction of the endocrine pancreas and in the development of diabetes. In other endocrine organs, there is little evidence for a role of NOX family enzymes, albeit some immunolocalization data exist , H 2 O 2 generation by thyrocytes was described by the early s 88 , , and an NADPH oxidase activity was quickly suggested to be the underlying mechanism The physiological role of DUOX in the thyroid is well studied and well understood.

Synthesis of thyroid hormones requires hydrogen peroxide for oxidation and incorporation of iodine into thyroglobulin. DUOX provides the hydrogen peroxide for this reaction. Despite the presence of a peroxidase homology domain in DUOX enzymes, the peroxidase reaction requires a separate enzyme, namely, the thyroid peroxidase. Biallelic inactivating mutations in the DUOX2 gene result in complete disruption of thyroid-hormone synthesis and are associated with severe and permanent congenital hypothyroidism, while monoallelic mutations are associated with milder, transient hypothyroidism DUOX2 sequence variants associated with mild hypothyroidism have also been described In contrast, islet cells appear to have a very low antioxidant capacity In both studies, an increased expression of these NOX isoforms in animal models of diabetes has been proposed , In line with this concept are observations that glucose toxicity can be prevented by antioxidants Gastric epithelium is able to generate ROS.

The traditionally held view is that sources of radicals in the gastrointestinal tract include mucosal xanthine oxidase and NADPH oxidase found in the resident leukocytes of the lamina propria This question has been addressed predominantly in guinea pig stomach, where ROS generation by gastric mucosa has been convincingly documented The responsible enzyme was initially suggested to be NOX2 based on activity and expression at the protein level ; however, more recent results based on mRNA suggest that it is rather NOX1 However, the expression of NOX1 in the stomach might be species specific.

In the mouse system, the expression profile of NOX enzymes in the stomach has, to our knowledge, not been studied. The understanding of the role played by NOX enzymes in the stomach is at a preliminary stage. In the guinea pig, NOX1 is suggested to be involved in the regulation of growth and apoptosis of gastric mucosal cells