WARNING: the foods we cook for Abby are safe for her, but not necessarily for everyone. Please confirm any ingredients are safe for you before using in your diet. Food Allergies can kill and the best policy is complete avoidance. Read this post for more info.

Sunday, March 31, 2013

Abby's deviled Eggs

Thankfully Abby can eat eggs if we buy the right ones. One of the things Abby has always loved is deviled eggs.

6 cold hardboiled eggs
2-3 heaping tablespoons of coconut yogurt(or homemade mayo or coconut sour cream)
1 tablespoon braggs apple cider vinegar
1 teaspoon fresh minced dill
1 dash mustard powder(mustard seeds grind well in a coffee bean grinder)
salt and pepper to taste.
paprika(we love simply organic brand spices)

Combine yolks and all the other ingredients in bowl. I find using a handmixer blends and whips the yolks nice and smooth. If these are too dry add a bit more yogurt or mayo or more vinegar depending on the flavor you prefer. I used a large tip wilton frosting tip and bag to pipe the yolks back in..finished with a quick dash of paprika.

Happy Easter!

The older the girls get the more pleasure I get putting together "baskets" for them each Easter. This year we broke tradition and went with "gift bags" but only because I found the cutest tissue paper ever and HAD to buy it!

I know they are getting a little old for baskets right? Not really! If anything as they get older they value that gift on Easter Morning a lot more. They understand the value of money and the cost of things. They understand that I am fiercely practical and random useless items are truly a gift from the heart when I buy them. Derek and I get a huge amount of joy from their disbelief that we insist on continuing this childhood tradition.

I remember loving all the little dust collectors. Those odd little tiny wood boxes stamped with cute art, or a decorative hard boiled egg holder that I promise will never be used in my house. Those silly adorable chicks shaped out of bits of pipe cleaners. I can step back in time when shopping for those "pretties." For a day I can be young again and simply enjoy seeing some cute little item that has no value besides that it is little, and cute and something as a teen I simply would have had to have!

Now it isn't just pretties, but a sharing of joy. Today they reach toward adulthood in that they now understand the deeper value and Derek and I get to step back and be young again and simply enjoy small, cheerful frivolous things.

We do miss the candy. I always made sure to fill their baskets with the candy they hated and I loved to ensure they shared. We miss seeing stale peeps stacked on the table for a week afterwards because who really eats them? This year I skipped the plastic grass and am regretting that just a little too- I am wondering if I will feel I am missing something if I am not vacuuming shreds of green grass for the next two weeks? Their gifts are a reflection of how they have grown and how we struggle to be young again.

Candy or not- each holiday only gets better as they get older. Today we will have fun, together.

Happy Easter!

Friday, March 29, 2013

More Americans on disability then food stamps and welfare combined..

Yet, the headlines in the news this week and every week is about which hollywood diva is pregnant, or which talk show host is tanking... even in the political arena not a peep about in a few short year there will be more disabled American's then capable tax paying Americans-

It is an EPIDEMIC!

When the graph shows more children with LD's, behavior and physical disabilities then not.

In 20 years who is going to be able to work? Abby is grateful to have the energy occasionally to shower on her own let alone hold a job or participate in keeping America on track...

Obamacare will cause some costs of private insurance to increase as much as 62 percent!!!

Seems like daily Drs. are opting to retire early instead being Governed by a government medical program that will no longer care for the individual.

If 1 out 50 children have autism, if 1 out of 4 American's(soon to be 2 out of 4 at the current rate) are too disabled to work- who is paying the bills while we rot in Government run nursing homes 20 years from now? Who will be left to care for us? To care for our sick children?

There are days I am beyond shocked that some how so many of our children are being destroyed one after another yet, not even a press conference..no government think tanks, no massive hunt to stop the epidemic- where is the CDC? Chasing the bird flu 3 countries away- guess they don't see an entire generation that will have more disabled then capable as an epidemic.

The only focus this Administration has taken on for future taxpayers is to get kids to eat more greens then cheese burgers. I agree with them but at this point some kale just isn't going to even come near fixing the catastrophe that is building before our very blind eyes.

As a parent I have to think about how my children will manage without us one day. No matter how much life insurance, no matter how much savings we diligently save I cannot save enough to insure she is cared for as I provide now.

Who will be there to care for all these sick children when they grow up?

Who will pay taxes?

When the next generation is under educated, disabled, who will pay the bills?

No one is trying to Prevent this epidemic from spreading- except a few parents. What is causing it? Why suddenly are two generations developing one illness after another? All these "new diseases" what caused them?

Do we as a Country just continue to hope if we close our eyes it will go away? It isn't.

If my kids cannot care for me or themselves, if your kids cannot care for me or you or themselves- who does that leave?

Wednesday, March 27, 2013

"Sugar" does a body good!

If you follow my blog, you know we mix "sugar" into what seems like everything! If there is a way to make a product into a dessert I tend to figure it out. In our family we found a couple generations ago, sugar just seems to help us feel better. Despite a few generations of dosing with sugar when ill, no diabetes in women and the few men that developed it were over 60 and had other health issues and very western diets. Again and again when Abby is crashing or flaring adding sugar seems to help her body "wake up" and get busy working again. If you have ever had an IV you know how wonderful you feel once you are "hydrated"- not just water in that IV!- Sugar,salt...

Now, we don't eat sugar 24/7 but we do eat more then traditionally recommended. With good reason.

Believe it or not, not all sugar is equal. Breast milk, baby formula, IV's, supplemental formulas- LOADED with sugar- Why? Because our bodies need sugar- ideally natural and raw and fructose is ideal- but in a pinch..of course there are exceptions and I don't recommend you start dosing with sugar against your Drs. recommendation- but for those who aren't sugar phobic- try a few new sugars for max benefit.

We are picky about which sugars though. Natural fructose, clean sucrose,pure coconut sugar, pure palm sugar, pure raw honey, and clean maple syrup(caution- just because maple is 100 percent it still can be "corned" from the de foaming agents used during processing.) We avoid date sugar because Abby reacts to dates and we avoid beet sugar because not only does she react to beets the vast majority of beet sugar is GMO.

Right now our latest favorite in sugar is Coconut Sugar-pure, clean and sweet. A very concentrated sweet if you buy it in a block. Surprise surprise, it isn't an empty sugar! With the baking I have done with it, it substitutes for granulated pretty well too!

A little information about why coconut sugar might just be a good thing to add to your diet:

LiveStrong What are the BENEFITS of Coconut Sugar?

Low Glycemic Index
The glycemic index is a numeric value given to a food to represent how fast it turns to sugar. Coconut sugar's glycemic index is approximately 35, which is considered very low. Low glycemic index foods can help balance and control health conditions such as diabetes, high cholesterol and obesity. Eating low glycemic may also protect your body against cardiovascular disease and hypoglycemia, which is a condition that causes the body to overproduce insulin and take too much sugar away from you, leaving you feeling weak, tired, irritable and always hungry. In fact, in a study published in the "American Journal of Clinical Nutrition," researchers from the University of Sydney in Australia showed that eating a low glycemic index diet reduced the risk of chronic diseases such as Type 2 diabetes, gallbladder disease, coronary heart disease and breast cancer.
High in Vitamins and Minerals
Coconut sugar contains the minerals calcium, magnesium, potassium, zinc, iron, copper, manganese, phosphorus and boron. Minerals are necessary for many body functions, such as muscle and bone growth, cell production, mental development and immune system and enzyme regulation. Because the body cannot produce minerals, it is imperative that you find them through food sources. Minerals are also necessary for vitamins to work efficiently in the body. Coconut sugar also contains 12 of the B vitamins, including thiamine, riboflavin, pyridoxine, nicotinic acid, biotin, pyridoxal and inositol. It is especially high in the vitamin inositol, which is necessary for nerve transmission, metabolism of cholesterol and the redistribution of fat within the body. In addition, inositol has been used to treat diabetes, depression and panic attacks.
Rich in Amino Acids
Coconut sugar contains 16 out of the 20 amino acids. This sugar is especially high in the amino acids glutamic acid, which makes glutamine, threonine, aspartic acid and serine. Glutamine is very important for metabolic function. According to the University of Maryland Medical Center, glutamine can also be effective for healing of wounds and injuries, building up the immune system, treating inflammatory bowel syndrome, helping with HIV/AIDS patients and possibly aid in the treatment of cancer.

Read more: http://www.livestrong.com/article/367337-what-are-the-benefits-of-coconut-sugar/#ixzz2Ol6Eeg8t

Clearly far from Empty! Vitamin B, 16 of 20 Amino Acids, magnesium, calcium, potassium .. and so much more! Did you catch that it is high in Glutamic Acid?

So throw out the corn crap but please add some pure coconut sugar.

Here is a quick link that I found interesting about lactic acid, Co2 and SUGAR! :-) if you want to read a little deeper into the BENEFITS of sugar-

Ray Peat Lactate vs Co2 in wounds, sickness and aging

Tuesday, March 26, 2013

Watermelon Sorbet

Despite my hesitation to try new foods with Abby right now, she is craving fruits and veggies. Not only does she need the nutrition but it is spring and knowing all the good veggies and fruits are coming makes you want them more! In the past she has done well with watermelon despite not doing well in general with many melons. I have cross reactive issues with melon and am reasonably convinced that is the issue with Abby as well. She ate some of the watermelon the other night, without any obvious ill effect. Of course eating it straight is perfect, but I can never resist turning anything into a dessert. Sorbet is perfect. Cold,smooth and not too sweet a nice refreshing finish to any meal.

First, a few good reasons to eat Watermelon via Natural News-

(NaturalNews) Who would have thought the common watermelon packed so much nutrition? The usual notion about watermelon is they are just water with taste, harmless, but not a good choice for nutritional value. Wrong! Watermelon has the most nutrition per calorie of common foods.

Organic watermelons are hard to find and pricey when found. But conventionally grown watermelons are not among the dirty dozen of the most heavily sprayed produce. Instead, they are actually among the fifteen most clean of non-organic fruits and vegetables.

What Watermelons Have to Offer

Besides tasting great and being low in calories because watermelon is mostly water, it is an excellent source of Vitamin C, which a major antioxidant. It has a high beta carotene concentration, thus offering a fair amount of vitamin A as well. Both beta carotene with vitamin A help support good eyesight and prevent glaucoma.

High intakes of combined beta-carotene and vitamin C have demonstrated, through clinical and scientific studies, a propensity for warding off various cancers and heart disease, reducing arthritis symptoms, and minimizing asthmatic breathing problems.

A surprise nutrient is vitamin B, especially B1 (thiamine) and B6 (pyridoxine). Thiamine is important for maintaining electrolytes and nervous system signal transmissions throughout the body. Pyridoxine is essential for enzymatic functions that convert food into cellular energy.

The meat or pulp of watermelons is usually pink or red. Those colors indicate the highest content of lycopene, an antioxidant lauded for its ability to greatly minimize cancer risks. From the Worlds' Healthiest Foods website (source below), "... lycopene has been repeatedly studied in humans and found to be protective against a growing list of cancers. These cancers now include prostate cancer, breast cancer, endometrial cancer, lung cancer and colorectal cancers."

Then there are the minerals of potassium and magnesium, which watermelons also offer abundantly. Potassium is important for cardiovascular health and brain health and helps the kidneys eliminate kidney stone forming calcium as well as assists with the body's fluid retention.

Magnesium is considered the master mineral. It is involved with over 300 cellular metabolic functions. It happens to be lacking in our diets because of our depleted topsoils. Magnesium deficiencies are directly or indirectly related to most of our population's poor health issues. Obvious symptoms are irritability, tension, sleep disorders, and muscular cramping. After that, it's heart attacks and other serious illnesses.

Learn more: http://www.naturalnews.com/029157_watermelon_nutrition.html#ixzz2OeJQxEfW

Watermelon Sorbet:

8 cups watermelon, seeds and rind discarded
1 cup Simple Sugar Syrup(2 parts sugar, 1 part water,heated till sugar is melted and then chilled)(We use Domino's brand sugar)
1/3 cup (juice of one lemon) fresh lemon juice(organic lemons, watch out for corn in the wax coating!)
dash or two of pink himalayan salt


Puree the watermelon and salt(salt helps release the liquid from the pulp and intensifies the flavor) in a food processor/blender. Strain the pulp and collect the juice. Measure 3 cups of the juice and place in a bowl. Add the Simple Sugar Syrup and lemon juice and stir well. Freeze in an ice cream maker according to the manufacturer's instructions.

Monday, March 25, 2013

Hot Cross Buns- Abby style.

Gluten free, dairy free, corn free..

For Abby we had to skip the normal candied peels and raisins and cinnamon. We used stewed figs and allspice instead. We just could not have Easter without Hot Cross Buns- these are a hit.

3 cups all purpose gluten-free flour
⅓ cup sugar
2 packages yeast
½ cup powdered sugar(corn free)
2 teaspoons guar gum (soy-lite, you can try a mix of ground flax and ground chia seed instead to be soy free and corn free)
1 teaspoon salt(I use Pink himalayan a good source of magnesium)
1 tablespoon allspice
1 cup warm water
¼ cup grapeseed oil or melted coconut oil
3 eggs, lightly beaten
1 teaspoon Braggs apple cider vinegar
1 cup diced stewed figs(black misson)

3 tablespoons melted shortening of choice, for brushing(palm or coconut oil )

1. Preheat the oven to 375 degrees. Lightly grease a 10-inch cake pan.
2. Mix flour blend, sugar, yeast, powdered sugar, guar gum, salt and allspice together.
3. Add water, oil, eggs and vinegar and beat for a few minutes till sticky and elastic
4. Mix in stewed diced figs(or raisins and candied peels if desired).
5. Scrape dough out onto a floured cookie sheet. Cut the dough into 8 or 9 pieces and gently shape each into a ball. Place one ball of dough in the center of prepared cake pan. Loosely arrange the remaining balls around it, leaving room for buns to rise. Brush the buns with melted shortening and cut an X into the top of each bun. Cover with a piece of plastic wrap and let rise in a warm, draft-free spot for 30 to 45 minutes(roughly doubled in size).
6. Place buns in preheated oven and bake for 30 to 35 minutes until golden brown. Remove from oven and cool on a rack. When the buns are cool, drizzle icing over the scored X
1½- 1 3/4 cups powdered sugar, sifted(and corn free)
2 tablespoons coconut yogurt plain or rice or coconut milk
1-2 teaspoon's fresh squeezed lemon juice
Whisk powdered sugar, yogurt or milk and lemon juice together until smooth. Add more lemon juice if icing is too thick

Saturday, March 23, 2013

Not our Favorite Month

I don't want to say we are "doubting" our choices, but March and April are historically difficult on Abby. With the tree pollen maxing out and grass pollen trailing quickly- it sure does a number on her.

For as much as I want to follow our schedule of trying "new" foods, or at least going back and trying some she previously did poorly with, we are on hold.

For the most part, we are insanely positive about Abby's health. In my family we have some terribly sick folks over the generations live into their 80's - some were worse off then Abby. When I think about their lives and how they managed I often chalk it up to "ignorance is bliss." "Knowing" the enemy that hangs over our house usually empowers us, but when Abby is struggling any little doubts tend to float to the surface. At times I really have wished for any other path, for blind faith, for ignorance- but you just cannot go back- no matter how uncomfortable, Abby and I and family must press forward.

Another teen that reminds me of Abby in many ways has been in and out of the hospital, he is very unstable right now and I am terrified for him, his family. The reality of how "unknown" these disease process's are, make me on one hand so incredibly grateful that with extreme diligence to how sensitive Abby's body is we have tailored her diet and environment to prevent further stressors. It is also frightening that something could go wrong with Abby's body that is out of our control.

Guess we can call it a reality check. A good time to decide if we have made good decisions this past year is when she is feeling her worst.

Yes, we feel strongly we have made all the right decisions. We know for a fact that the changes we have made have done what no medicine or medical intervention could do. Every choice we make however has consequences, cause and effect. Sometimes even the right decisions can have a lofty price.

When I see her struggle to find the energy to wake up, to watch her struggle out of bed with pain written all over face, to know her kidneys are in horrid pain, to know her gut is not in the mood for anything new, when she gives me the nights headlines of struggling to breath, or odd tingles, pain, reactions.. I do wish with all my heart I knew more- that I had some book to refer to that had even half the answers. Despite her struggles she always manages to overcome. Even miserable she is more worried about the teen in the hospital then herself. I find her reassuring me that we need to just chill until the tree pollen slows.. that she has always had bad days or weeks and with time and patience she AlWAYS feels better.. anytime her body cuts her some slack she runs with it. Despite "seeing" her misery written on her face you would never know from her laugh or her words- tough kid.

Project Elimination worked because we tailored it to Abby's very specific needs. Each time I have to analyze new changes and make decisions on which direction we need to go next I feel paralyzed. I know without a doubt that the wrong food, temperature, activity level could cause her to slip- yet, it feels like I am playing that game show of having to pick between door 1, door 2, door 3 or door 4 - at best each decision is heavily laced with instinct. I would prefer picking based on facts- but for Abby we don't have enough facts yet to gamble on predictable outcomes.

Really what Abby is dealing with this month is nothing compared to so many. We aren't talking life and death, but we are talking about additional damage that adds up and slows the healing- it is so frustrating.

Today we are going to remind ourselves, March is just a bad month. Damage control. Most importantly when I compare what I see this year to how she was last year, or the year before? She looks great in comparison-

When you deal with the unknown, you cannot afford to have blind faith. You or your Dr. do not have a diagnostic and treatment guide book, there are no certain outcomes. Very much like when way way way back EVERYONE thought the world was flat- a few saw it differently-

No one then could believe the world was round just like no one believes there is a way right now to beat this - fact is there is a way to beat we just don't know what that is yet.

Thursday, March 21, 2013

Go MitoAction!

This is a brilliant use of time and resources on the part of MitoAction!

MitoAction hosts Mitochondrial Disease Clinical Conference!

In an ongoing effort to improve mitochondrial disease patient care, MitoAction, in collaboration with the organization’s Medical Advisory Committee, will host the 2013 Mitochondrial Disease Clincial Conference on May 4, 2013 at Hotel Marlowe in Cambridge, MA.
(PRWEB) March 20, 2013
In an ongoing effort to improve mitochondrial disease patient care, MitoAction, in collaboration with the organization’s Medical Advisory Committee, will host the 2013 Mitochondrial Disease Clincial Conference on May 4, 2013 at Hotel Marlowe in Cambridge, MA.
The conference will offer physicians and specialists who are not mitochondrial experts an opportunity to understand more about identifying mitochondrial diseases, managing adults and children with mitochondrial disorders, and assisting patients with treatment options and coordinated care. Expert faculty will provide the most clinically relevant information with the intention of helping to educate healthcare providers about specific strategies for helping the mitochondrial disease patient. The conference has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Tufts University School of Medicine (TUSM) and MitoAction.

Read more: http://www.newstimes.com/business/press-releases/article/MitoAction-hosts-Mitochondrial-Disease-Clinical-4369282.php#ixzz2OBOgtZrY

The fact is, we don't have enough Mito Specialists to go around. Fact is, as Mito Medicine evolves each Mito Specialist is developing and focusing on certain aspects of Mitochondrial Disease. Too many stories of Mito Patients being turned away, or Suspected Mito patients not getting care.. Not blaming Mito Specialists-they only have so much time,money and passion. Each focusing on certain types of Mito will be tremendously helpful down the road but fact is that many in Mito Population are not receiving adequate care. Also, we have seen in the past year that a lot of research about Mito is bleeding into Cancer, parkinson's, Dementia- which means less research on Primary Mitochondrial Disease patients- which is just making things harder for the patients and Drs. I googled Mitochondria and Cancer- WOW they are funding and researching like mad! Cancer is a better funded disease hands down- eventually the trickle down research will apply to the straight up Mito patients, but not today.

Family Drs and Peds are on ground zero. Too many have no idea about what Mito is.. if they do know often is a very generalized and outdated view. They have not been taught that Mitochondrial Disease is so incredibly individual in how it presents. They have not been taught that each patient even with the same gene mutation needs completely individualized care,may exhibit completely different symptoms and often have very different perspectives on how to treat their disease. I can honestly say that the family Drs. and Peds we have met over the years tend to be far more willing to help then any specialist Abby has seen but they don't know how to help. The only choice they have is to refer us.

Even if you find a Family Dr. who has treated another Mito patient, odds are it doesn't prepare them to treat you or yours- they have not been taught that more often then not what patient A needs, won't be what patient B needs.

I think between patient education and educating the Drs. who are in the trenches we can eliminate much of the reliance on Specialists. This would make care much easier for many Mito patients and better care. It would also allow Mito Specialists to focus on research instead of patient care- which benefits the Specialist, the family Dr. and most importantly the patient.

Another Caramel-

(leftover rice,dash of salt,sprinkle of cardamon, coconut milk(2 to 1 with cooked rice) cook on stove till starts to thicken and instead of sugar I mix caramel in while it is still hot!)

With all of the substitutions we make when recreating recipes for Abby, we have had hit or miss results for caramel. It always tastes great it is usually a texture issue or not consistent results. With this recipe we are on the fourth batch and perfect every single time. Smooth- and Abby says it the closest to real caramel we have tried yet. We have tried an obscene amount of recipes at this point and this is a keeper. I need to make another batch of homemade cane syrup and see if it works as well as the Lyle's purely from a cost standpoint. The advantage of the Lyle's is it has that buttery taste that we miss since we cannot use dairy products. Worth the cost. It hasn't been used by many in the corn allergy community, but so far Abby has not reacted.


grapeseed oil, for baking sheet
1 3/4 cups coconut cream (I try very hard to use only the cream from the top of the coconut milk)
2 1/4 cups sugar(domino's)
6 tablespoons Palm Shortening(Spectrum)
1 1/4 cups Lyle's Golden Cane syrup( not corn allergy population tested)
1/2 teaspoon coarse salt(we use pink himalayan)
1/2 teaspoon vanilla extract(we have skipped the vanilla and it was fine, but use homemade to avoid corn)


Lightly brush bottom and sides of a 8x8(or 9x13 but we like thick pieces)inch pan with oil. Line with parchment, leaving a 2-inch overhang on long sides; lightly brush parchment with oil.

Bring coconut cream, sugar, palm shortening, and Lyle's Golden cane syrup to a boil in a large saucepan over high heat, stirring until sugar dissolves. Reduce heat to medium-high; cook, stirring occasionally, until caramel reaches 248 degrees on a candy thermometer, about 15 minutes.

Immediately remove caramel from heat, and stir in salt and vanilla. Pour caramel into grease and lined(believe me you need it lined! I have tried just greasing the pan and it sooo hard to try to cut and remove the caramel) pan of your choice, and let stand, uncovered, at room temperature at least 8 hours and up to 1 day.

Monday, March 18, 2013

Lentil Lemon Snacking Cake(Gluten free, corn-free, soy-free, dairy-free)

(The color is brown because I used the brown teff flour in my gf flour blend)

Chickpea's are on rotation- and we are trying Lentils. We had to rotate the chickpea's as too much of anything always backfires with Abby and chickpea's are so essential to help her nutrition I could not stand the thought she may develop a reaction. Last year at the beginning of project elimination we tried lentils and it was not a good food for her. At the time her gut was still so wrecked from all the abuse of eating allergens and everything she really did not digest much well at all. When I introduced chickpeas we soaked,rinsed, soaked then cooked the heck out of them and pureed them smooth. Anything to make it a little easier for her body to process- we are hoping the same effort with the lentils is a go!

1 cup all-purpose gluten free flour
3/4 granulated sugar(domino's)
1/2 teaspoon baking powder(Hains or homemade)
1/8 teaspoon baking soda
Pinch of salt
1/2 cup coconut yogurt(Homemade,no store bought coconut yogurt is clean enough of corn for her to tolerate).
1/4 cup grapeseed oil
2 teaspoon grated lemon zest(Be warned! Often lemons are coated in wax or other products that may contain corn, some people in the corn allergy population react strongly to the various treatments- caution with organics as well)
1/3 cup fresh squeezed lemon juice
1 large egg
1/2 cup lentil puree( well soaked,well cooked lentils, pureed in the Ninja)
Garnish: Confectioners’ sugar(corn-free or homemade)

Instructions Preheat to 350 degrees.

You know the drill- mix all the "wet" ingredients including lentil puree, lemon zest, and coconut yogurt till well blended. Add the rest of the ingredients and mix till just smooth.

Pour batter into well greased 8 x 8 pan. Bake for 35-40 minutes or until a knife comes out clean or mostly so..

Allow to cool and sift top with corn-free powdered sugar. We preferred this cake chilled.(Honest, you cannot taste those lentils!)...

This morning we aren't sure about the lentils yet, nothing obvious but she has been flared with all the tree pollen so we are going proceed with caution- sure tasted great though!

Article: Mitochondrial dysfunction increases allergic airway inflammation

Stumbled across this one(and another great one about vitamin D deficiency and how low levels might effect mitochondria) like most of these journal articles -long winded, but worth the read.

US National Library of Medicine
National Institutes of Health

Journal List >NIHPA Author Manuscripts >PMC3028535

J Immunol. Author manuscript; available in PMC 2011 January 27.
Published in final edited form as:
J Immunol. 2009 October 15; 183(8): 5379–5387.
Published online 2009 September 28. doi: 10.4049/jimmunol.0900228
PMCID: PMC3028535

Mitochondrial dysfunction increases allergic airway inflammation

Leopoldo Aguilera-Aguirre, MSc,*† Attila Bacsi, PhD,* Alfredo Saavedra-Molina, PhD,† Alexander Kurosky, PhD,‡ Sanjiv Sur, MD,§ and Istvan Boldogh, PhD*,2
Author information ► Copyright and License information ►
The publisher's final edited version of this article is available free at J Immunol
See other articles in PMC that cite the published article.
Go to:
Pollen grains and subpollen particles contain NAD(P)H oxidases and shown to induce oxidative stress in the airway epithelium; however, the role of mitochondrial dysfunction induced by them has not been investigated on airway inflammation. Our results show that exposure of airway epithelial cells to ragweed pollen extract (RWE) induced oxidative modifications to NADH dehydrogenase (ubiquinone) Fe-S protein (NDUFS) 1 and NDUFS2 in mitochondrial respiratory complex I, as well as ubiquinol-cytochrome c reductase core protein I (UQCRC1) and II (UQCRC2) in complex III. Respiratory chain-associated proteins, the 75-kDa glucose-regulated protein, heat shock protein 70, heat shock protein 60, citrate synthase and voltage-dependent anion selective channel 1 were also damaged. Exposure of cells to RWE induced mitochondrial dysfunction as shown by increased H2O2 release from respiratory chain complex III. Mitochondrial dysfunction induced by antisense oligonucleotides to UQCRC2 increased mitochondrial ROS generation in lung epithelial cells. Most importantly, mitochondrial dysfunction in airway epithelium of sensitized Balb/c mice prior the RWE challenge increases the RWE-induced accumulation of eosinophils, mucin levels in the airways and bronchial hyperresponsiveness. A decrease in UQCRC1 expression did not significantly alter cellular ROS levels and parameters of airway inflammation. Based on these observations preexisting mitochondrial dysfunction induced by oxidant environmental pollutants and/or pollen grains exacerbate antigen-driven allergic airway inflammation. These data also imply that mitochondrial defect could be a risk factor and may be responsible for severe allergic disorders in atopic individuals.

Keywords: mitochondria, allergy, inflammation, lung
Go to:
It has been demonstrated that pollen grains and subpollen particles themselves generate reactive oxygen species (ROS)3 via their intrinsic NAD(P)H oxidases (1, 2). ROS generated by these pollen enzymes induce profound oxidative stress in the lungs within minutes after exposure (3). Inactivation of pollen NAD(P)H oxidases or elimination of oxygen radicals generated by them decreases the allergic inflammation in sensitized mice after pollen exposure (3, 4). Environmental factors including ozone, diesel exhaust particles, and tobacco smoke, as well as respiratory virus infections, which are known to increase the ROS production exacerbate allergic inflammation in asthma (5). Excessive levels of ROS can trigger signaling cascades and/or cause cellular damage through the oxidation of macromolecules, even in the mitochondria (6, 7). Indeed, it has been shown that ozone exposure, tobacco smoke and other environmental oxidant pollutants, induce oxidative damage to mitochondria in the airways (8–10). Based on these observations and the exclusively maternal transmission of mitochondria in humans (11), as well as a predominant maternal influence on atopy and asthma susceptibility, we proposed that oxidative damage-caused mitochondrial dysfunction contributes to underlying molecular processes in the development of allergic airway inflammation. We tested this hypothesis in a mouse model allergic asthma.

Herein we demonstrate that exposure to ragweed pollen extract induces oxidative modification of mitochondrial respiratory chain complex I and complex III resulting in mitochondrial dysfunction. We have identified that oxidative damage primarily to ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) elevates production of mitochondrial ROS. Importantly, alteration of UQCRC2 expression level prior to allergen exposure significantly enhances antigen-induced airway inflammation and bronchial hyperresponsiveness in experimental animals. These results suggest that preexisting mitochondrial dysfunction inflicted by environmental oxidant pollutants intensifies antigen-driven allergic airway inflammation and could be a risk factor for development of atopic allergic disorders in susceptible individuals.

Go to:
Cell cultures

Human alveolar epithelial cells (A549) and mouse lung adenoma cells (LA-4) were obtained from American Type Cell Collection (ATCC) and cultured in Ham’s F-12 medium supplemented with 10% (A549) or 15% (LA-4) heat-inactivated FBS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere with 95% air and 5% CO2. Mitochondrial DNA-depleted cells (ρ0) were developed as we had previously described (12). Briefly, A549 cell cultures were maintained in complete growth medium supplemented with 50 ng/ml ethidium bromide for >60 population doublings. Respiration-deficient cells became pyrimidine auxotrophs, and hence medium was supplemented with uridine (50 μg/ml) and sodium pyruvate (120 μg/ml) (13). Depletion of mitochondrial DNA was confirmed by Southern blot hybridization as previously described (14).

Measurement of intracellular ROS levels

Cells in suspension were loaded with 5 μM 2’,7’-dihydro-dichlorofluorescein diacetate (H2DCF-DA) for 15 min at 37 °C. After removing excess H2DCF-DA, cells were treated with 100 μg/ml of RWE and the changes in DCF fluorescence were determined at different time points by flow cytometry (BD FACS CantoTM Flow Cytometer, Benton Dickinson, San Jose, CA). Each data point represents the mean fluorescence of 12,000 cells, from three or more independent experiments. Alternatively, in parallel experiments cells at 70% confluence were loaded with 50 μM H2DCF-DA on 24-well plates (Costar, Corning, NY). Changes in fluorescence intensity in mock-treated and RWE-treated cells were measured using an FLx800 Microplate Fluorescent Reader (Bio-Tek Instruments, Winooski, VE) at 488 nm excitation and 530 nm emission wavelengths.

Mitochondria isolation and purification

Mitochondria were isolated and purified as we had previously described (12, 15). Briefly, cell pellets were incubated in a hypotonic buffer A (220 mM mannitol, 70 mM sucrose, 2 mM MOPS and 1 mM EGTA, pH 7.4) containing protease inhibitor cocktail (catalog No. P8340, Sigma-Aldrich, St. Louis, MO). Cell suspensions kept in ice bath were sonicated with a Branson sonifier by 4 pulses of 20% power and cell homogenates were centrifuged for 10 min at 4700 × g. Mitochondria were sedimented from supernatants by centrifugation at 7168 × g. Pellets were resuspended in buffer B (220 mM mannitol, 70 mM sucrose, 2 mM MOPS, pH 7.4) and centrifuged at 9072 × g. Crude mitochondrial solutions were layered on discontinuous sucrose gradients (1.5, 1.0 and 0.5 M sucrose in 10 mM MOPS and 1 mM EDTA, pH 7.4) (16) and ultracentrifuged for 1.5 h at 82705 × g (SW28 rotor, Beckman Coulter, CA). All centrifugation procedures were carried out at 4°C. The band containing mitochondria was removed and washed in 10 times volume of buffer B. Mitochondrial pellets were resuspended in buffer B containing protease inhibitor cocktail (Sigma) and kept in −80°C.

Isolation of mitochondrial respiratory complexes

Mitochondrial respiratory complexes (MRC) were isolated by blue native PAGE as previously described (17). Briefly, mitochondria (200 μg protein) were solubilized in 50 mM imidazole-HCl, 750 mM 6-aminocaproic acid (pH 7.0), and dodecyl maltoside (at a detergent-protein weight ratio of 1:4) for 30 min at 4oC and centrifuged at 16,100 × g. The supernatant was supplemented with Coomassie Brilliant Blue G-250 (at a dye-protein weight ratio of 1:4) and 10% glycerol and MRC complexes were electrophoresed in a 5–12% blue native PAGE (18). Mitochondrial respiratory complex bands were excised from the gel and incubated in a denaturing buffer (6% SDS, 150 mM 2-mercaptoethanol) and then placed on a 10% SDS-PAGE to separate individual proteins. To confirm the identification of MRC proteins, a MS601 MitoProfileR Human Total OXPHOS Complexes Detection Kit (Mitosciences, Eugene, OR) was used according to the manufacturer’s protocol.

Detection of oxidized proteins

Changes in oxidized protein levels were determined using an Oxyblot kit (Chemicon/Millipore, Temecula, CA) according to the manufacturer recommendations. Briefly, proteins were derivatized with 4-dinitrophenylhydrazine (DNPH) for 15 min followed by incubation at room temperature with a neutralization buffer (Chemicon/Millipore). Derivatized proteins were electrophoresed on a 10% SDS-PAGE and blotted on Hybond PVDF membranes (Amersham, Piscataway, NJ). Blots were blocked with 5% non-fat dry milk (blocking buffer) in Dulbecco’s PBS containing 0.05% Tween 20; (PBS-T) for 3 h and incubated with anti-DNP primary antibody (1:150) (Chemicon/Millipore) overnight at 4oC. In selected experiments, blots were incubated with primary antibody to 4-hydroxynonenal-protein adducts (1:300) (Oxis International Inc., Foster City, CA). In control experiments, blots were analyzed for components of the mitochondrial respiratory chain complexes using OXPHOS monoclonal antibody cocktail (1:200) (Mitosciences). After three washes with PBS-T, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:300) (Amersham, Piscataway, NJ). Immunocomplexes were visualized by chemiluminescence using the ECL kit (Amersham).

Protein identification

Protein bands were excised from Coomassie Blue stained gels, digested with trypsin, and subjected to mass spectrum analysis using a Model 4800 MALDI-TOF/MS analyzer (Applied Biosystems, Foster City, CA). Proteins were identified using the Swiss-Prot database and the Mascot algorithm as we reported previously (19). Expected values were considered significant when E ≤ 0.01 (20). MS analyses were conducted by the Biomolecular Resource Facility at UTMB.

Amplex Red Assay

Release of H2O2 from isolated mitochondria was measured by AmplexR Red (10-acetyl-3,7-dihydroxyphenoxazine; Molecular Probes) assay as we previously described (12). Briefly, mitochondria (100 μg/ml) were suspended in 50 μl (per well) reaction buffer and incubated with 0.25 U/ml AmplexR Red and 1 U/ml of HRP at 25°C for 30 min. The changes in fluorescence intensity were measured using a microplate reader (SpectraMass M2, Molecular Devices, Sunnyvale, CA) at 530/590 nm. The addition of catalase (400 U/ml, Sigma Inc), which catalyzes the decomposition of H2O2 to water and oxygen, decreased fluorescence signals by 90%. As a positive control, increasing concentrations of H2O2 (0–400 pmol) were used.

Mitochondrial quality was assessed by respiratory control ratio as previously described (21). Mitochondrial respiratory state 4 (ST4, resting state) was determined by oxygen consumption rate in the presence of respiratory substrates (5 mM glutamate/5 mM malate or 10 mM succinate/1μM rotenone). Respiratory state 3 (ST3, active state) was determined by oxygen consumption rate in the presence of respiratory substrates plus ADP and Pi. Respiratory control ratio was calculated as the ratio of ST3/ST4. Mitochondrial suspensions showing respiratory control ratio values > 2.5 were used for further experiments.

Down-regulation of target genes by antisense oligonucleotides

Antisense oligonucleotides (ASO) were designed by siDESIGNR Center on-line software (Dharmacon RNAi Technologies, Lafayette, CO) using the sequence database of the National Center for Biotechnology Information. The phosphorothioate protected ASO were synthesized by Integrated DNA Technologies (Coralville, IA). Down-regulation of target gene expression was evaluated by real time-RT PCR. Total RNA was isolated using an RNAqueous kit (Ambion, Austin, TX). A template of cDNA was synthesized using SuperScriptTM III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, CA). Real time PCR primers for UQCRC1 (Cat # PPM41408A) and UQCRC2 (Cat # PPM24606A) (SABiosciences, Frederick, MD) were used to analyze expression levels of these genes. To analyze transfection efficiency, Texas Red labeled ASO was added or transfected into LA-4 cells. Four h later cells were dried, fixed in acetone/methanol (1:1) and stained with 10 ng/ml of 4,6'-diamidino-2-phenylindole (DAPI). Cells were mounted on microscope slides and analyzed using a Nikon Eclipse TE 200 fluorescent microscope attached to a Photometrix CoolSNAP Fx CCD digital camera and MetaMorph software (version 6.09; Universal Imaging Corp.). Down-regulation of target genes by ASO was calculated based on percent of transfected cells and levels of PCR amplification. For down-regulation of UQCRC1 and UQCRC2 the following ASO were selected: UQCRC1; 3’-t*c*t*tgcaccagaaact*a*a*t and UQCRC2; 3’-g*g*t*acgacgataacaa*c*g*t (*phosphorothioate).

Sensitization and challenge of animals

BALB/c mice were purchased from Harlan Sprague-Dawley (San Diego, CA). All animal experiments were performed according to the National Institutes of Health Guide for Care and Use of Experimental Animals and approved by UTMB Animal Care and Use Committee. Eight-week old female mice were sensitized with RWE as previously described (3). Briefly, mice received two intraperitoneal administrations (days 0 and 4) of endotoxin-free (150 μg per animal) RWE (lot 34868; Greer Laboratories (Lenoir, NC) mixed with alum adjuvant (Pierce Laboratories, Rockford, IL) at a ratio 3:1. RWE-sensitized mice received intranasally 10 μg of antisense dissolved in 60 μl PBS at 36, 24 and 12 h prior to RWE challenge. Control mice received same volumes of PBS. On day 11, mice were challenged intranasally with RWE (100 μg dissolved in 60 μl PBS).

Assessment of UQCRC1 and UQCRC2 expression in the lungs

Levels of UQCRC1 and UQCRC2 proteins were analyzed by fluorescent microscopy. Briefly, lung sections were blocked for 30 min with rabbit non immune serum (1:100). After two washes with PBS-T, slides were incubated with primary antibodies to UQCRC1 (1:200) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) and UQCRC2 (1:200) (Novus Biologicals, Littleton, CO) as well as to cytochrome c oxidase subunit IIb (Cox IIb; Molecular Probes) for 60 min at 30°C. After two washes with PBS-T, cells were incubated with FITC-conjugated (Santa Cruz Biotechnology) or Texas Red-conjugated (Leinco Technologies, Inc., St. Louis, MO) F(ab’)2 secondary antibodies (1:200) for 60 min at 37°C. After a wash with PBS-T, cells were counterstained with 10 ng/ml of DAPI, and mounted using DakoCytomation (Carpinteria, CA) mounting medium. Sections were analyzed using a Nikon Eclipse TE 200 fluorescent microscope attached to a Photometrix CoolSNAP Fx CCD digital camera and MetaMorph software (version 5; Universal Imaging Corp.).

Differential cell counts

Three days after RWE challenge mice were euthanized and bronchoalveolar lavage fluids (BALF) were collected. Trachea was cannulated and lung lavage was performed by two instillations of 0.6 ml ice cold PBS. The BALF samples were centrifuged (800 × g for 5 min at 4oC), and the resulting supernatants were stored at −80 oC for further analysis. Total cell counts in BALF were determined from an aliquot of the cell suspension. Differential cell counts were performed on cytocentrifuge preparations (Shandon CytospinR 4 Cytocentrifuge, Thermo Scientific, Waltham, MA) stained with Wright-Giemsa, in a blind fashion by two independent researchers counting 200 cells from eachanimal.

Assessment of mucin levels

MUC5AC levels in the BALF were assessed by ELISA as previously described (22). Briefly, 96-well plates were coated with serially diluted BALF in coating buffer (50 mM Na2CO3, 50 mM NaHCO3) and then primary mouse anti-human MUC5AC antibody (1:16000) (clone 45M1, Lab Vision, Fremont, CA) was added. Binding of primary antibody was detected by peroxidase-conjugated goat anti-mouse secondary antibody (1:5000) (Amersham) and a peroxidase substrate, tetramethyl benzidine (eBioscience, San Diego). Changes in absorbance were measured at 450 nm using a SpectraMax 190 ELISA plate reader (Molecular Devices, Sunnyvale, CA). All incubations were done at 37°C.

Evaluation of allergic inflammation by histology

After bronchoalveolar lavage, the lungs were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned to 5 μm and stained with haematoxylin and eosin. Perivascular and peribronchial inflammation were evaluated by a pathologist in a blinded fashion to obtain data for each lung. Mucin producing cells were assessed by periodic acid–Schiff staining of formalin-fixed, paraffin embedded lung sections. The stained sections were analysed as above and representative fields were photographed with a Photometrix CoolSNAP Fx camera mounted on a NIKON Eclipse TE 200 UV microscope.

Airway hyperresponsiveness

Changes in pause of breathing (Penh) as an index of airway obstruction were measured by barometric whole-body plethysmography (Buxco Electronics Inc, Troy NY) (23). Bronchial hyperreactivity was evaluated using the methacholine challenges (24). Briefly, mice were placed in a Buxco chamber, allowed to acclimate for 5 min and then exposed for 3 min to nebulized saline. Subsequently, mice were exposed to increasing concentrations (0, 6.25, 12.5, 25 and 50 mg/ml) of nebulized methacholine (Sigma) in saline. The median size of the aerosol doplets ranged between 1 and 4 μm (manufacturer’s specification). Bronchopulmonary resistance was expressed as enhanced pause = [(expiratory time/relaxation time) − 1] × (peak expiratory flow/peak inspiratory flow). The flow signals and the respiratory parameters were calculated using the Biosystem XA program (Buxco Electronics).

Statistical analysis

Data are presented as mean ± standard error of the mean. Mice were randomly grouped (group size, 4 to 6). All experiments were repeated 3 to 5 times. Statistical analysis was performed using the Student's t-test or ANOVA, followed by post hoc tests: Bonferroni’s (samples with equal variances) and Dunnett’s T3 (samples with unequal variances) with SPSS 14.0 software. Differences were considered to be statistically significant at P < 0.05.

Go to:
Exposure to pollen extract induces oxidative damage to mitochondrial respiratory chain proteins

Pollen grains or their extracts induce oxidative stress in cultured cells and in the airway epithelium as well as conjunctiva of challenged experimental animals (1, 3). Here, we show that addition of RWE to human airway epithelial cells (A549), after an initial oxidative burst, resulted in a sustained increase in the intracellular ROS levels. At 6 h after RWE exposure, ROS levels decreased to basal values as shown in Figure 1. On the other hand, in mitochondrial DNA-depleted cells (A549ρ0) the oxidative burst was transient and lasted for approximately 1 h (Fig. 1). Diphenylene iodinium (DPI; 100 μM), a NADPH oxidase inhibitor (25), abolished the changes in ROS levels in both A549 and A549ρ0 cells (Fig. 1). Together, these results suggested that RWE-exposure caused oxidative modifications to mitochondrial respiratory chain resulting in elevated ROS generation from mitochondria.

FIG. 1
Exposure of cells with functional mitochondria results in sustained increase in the intracellular ROS levels. Airway epithelial cells (A549) cells and mitochondrial DNA depleted A549ρ0 cells were loaded with H2DCF-DA and treated with 100 μg/ml ...
To test this hypothesis, we isolated and purified mitochondria from RWE-treated and control cells. First, overall changes in modified protein levels were analyzed, and as shown in Figure 2, a notable increase in carbonylated protein levels was observed in mitochondrial lysates from RWE-treated cells, but not from mock-treated cells. Next, the MRC complexes were isolated by blue native PAGE (Fig. 3A), their DNPH-derivatized proteins were separated in a 10% SDS-PAGE and damaged proteins were visualized by anti-DNP antibody (Fig. 3B). Levels of carbonylated proteins were increased in mitochondrial respiratory complexes and complex-associated proteins from RWE-treated cells compared to those from mock-treated ones (Fig. 3B). In complex I, anti-DNP antibody did not react with any DNP-derivatized proteins from mock-treated cells; while, it detected 4 carbonylated proteins from RWE-treated ones (Fig. 3B, right panel, 1, 2, 3, 4). In complex III, 3 protein bands appeared, as consequences of RWE treatment (Fig. 3B, right panel, 5, 6, 7). In the case of complex IV, only quantitative differences in the levels of carbonylated proteins were observed (Fig. 3B). Due to RWE-exposure, levels of carbonylated proteins further increased and an additional 31-kD protein was oxidatively damaged in complex II (Fig. 3B, right panel, 14). Furthermore, we observed elevated levels of 4-hydroxynonenal-protein adducts from RWE-treated cells (data not shown). Their patterns were similar; however, their abundances were less pronounced than those of carbonylated proteins shown in Fig. 2A.

FIG. 2
Treatment of epithelial cells with RWE increases the levels of carbonylated proteins in mitochondria. Mitochondria isolated from mock-treated and RWE-exposed (100 μg/ml) cells were purified (Materials and Methods). Mitochondrial lysates were DNPH ...

FIG. 3
Detection and identification of carbonylated mitochondrial respiratory complex proteins. A, Mitochondria isolated from mock-treated and RWE-exposed (100 μg/ml) cells were purified. Equal amounts of mitochondrial lysates were loaded and MRCs were ...
MALDI-TOF/MS analysis of oxidatively-damaged proteins identified NADH dehydrogenase (ubiquinone) Fe-S protein (NDUFS) 1 and NDUFS2, both of them are parts of the catalytic core of the complex I (Table 1, Fig 3). Ubiquinol-cytochrome c reductase core protein I (UQCRC1) and UQCRC2, core components of the complex III, were also identified among the damaged proteins (Table 1, Fig 3). We did not find carbonylated electron transport proteins in either complex II or IV. In all experiments, several accessory proteins, co-migrated with the respiratory chain complexes during the blue native PAGE, were also damaged. These proteins include 75-kDa glucose-regulated protein (GRP75; associated with complex [C] III), heat shock protein (HSP) 70 (CI, CII, CIII, CIV) HSP60 (CII, CIV), citrate synthase (CISY; CII) and voltage-dependent anion selective channel 1 (VDAC1; CII) (Table 1, Fig 3).

Table 1
Identification of oxidatively damaged protein
Pollen extract triggers ROS production from mitochondrial respiratory chain complex III

To analyze the functional consequences of damage to respiratory complexes, we determined the levels of ROS released from isolated mitochondria from RWE-treated and control cells. The primary mitochondrial reactive oxygen radical is the superoxide anion, which is rapidly converted to H2O2 by enzymatic or non-enzymatic pathways (26). Mitochondria isolated from RWE-treated cells produced significantly higher amount of H2O2 than those from mock-treated cells (Fig. 4A). In control experiments cells were exposed to heat-inactivated RWE (3) or they were treated with RWE in the presence of DPI (100 μM) or antioxidant (N-acetyl-L-cysteine; 10 mM, 3-h pretreatment). The H2O2 productions of mitochondria from these cells were close to the basal levels. These data are in line with the notion that oxidative mitochondrial damage leads to elevated ROS production (27). Complex I (rotenone; 10 μM) or complex II (3-nitropropionic acid; 3-NPA, 3 mM) inhibitor significantly decreased mitochondrial H2O2 production (Fig. 4B). The combined use of rotenone and 3-NPA decreased the H2O2 production nearly to the basal level (Fig. 4B), suggesting that electron flow from complex I and complex II chain is required for the increased ROS generation. Stigmatellin, which inhibits the Qo site of complex III at low concentration (0.06 μM) (28), abolished the RWE-induced mitochondrial H2O2 generation, while antimycin A (10 μM), an inhibitor of the electron transfer from cytochrome b to ubiquinone (12), further increased it, suggesting that complex III is most likely the source of the released ROS (Fig. 4B). Together, these observations indicated that oxidative damage to complex I proteins either did not result in increased ROS generation or ROS were released into the mitochondrial matrix where they were eliminated by antioxidants. These data further supported that in RWE-treated cells complex III was the major site of ROS released into the inner membrane space of mitochondria altering intracellular oxidative stress levels.

FIG. 4
Mitochondria isolated from RWE-treated cells released higher amounts of H2O2 than mitochondria isolated from mock-treated cells. A, Pretreatment of cells with antioxidant (NAC) as well as physical (heat-treatment, RWEH) or chemical (DPI) inactivation ...
Down-regulation of UQCRC2 mimics its oxidative damage

Mitochondrial proteins, damaged upon oxidative insult, change their function and oxidized proteins are known to accumulate during various pathophysiological states associated with oxidative stress (29). Because of the increased H2O2 release from complex III after RWE exposure, we focused our investigations on UQCRC1 and UQCRC2 in these processes. To mimic the altered/decreased function induced by oxidative damage to these proteins, we down-regulated them at RNA level using antisense oligonucleotides (ASO) (both UQCRC1 and UQCRC2 are encoded by nuclear genes). The efficiency of down-regulation at RNA levels were determined by quantitative RT-PCR. Results corrected by transfection efficiency of cell cultures showed that ASO down-regulated expression by more than 80% (Fig. 5A). Due to varying percentage of efficiently transfected cells (determined by Texas Red-labeled ASO) we used in situ microscopic analysis to assess changes in cellular ROS levels. Microscopic images indicated that only cells with red fluorescence (Texas Red) showed increased DCF signal (green fluorescence) in UQCRC2 transfected cultures (Fig. 5B). The increased DCF fluorescence was seen at both 1 and 3 h after loading the cells with H2DCF-DA, which is consistent with sustained ROS generation shown in Figure 1. In similar experiments UQCRC1 transfected cells showed no increased DCF fluorescence (data not shown). Taken together, these results suggested that down-regulation of UQCRC2 increased mitochondrial ROS levels.

FIG. 5
Down-regulation of UQCRC2 increases cellular ROS levels. A, ASO to UQCRC2 down-regulated its expression in LA-4 cells by more than 80% (Materials and Methods). B, Increased ROS levels in cells efficiently transfected with ASO to UQCRC2. Cells were transfected ...
Oxidative damage to UQCRC2 in the airways increases allergic inflammation and bronchial hyperresponsiveness

Next, we investigated whether the preexisting oxidative damage to UQCRC2 exacerbates allergic airway inflammation. Sensitized mice were repeatedly treated with the corresponding ASO (3-times, 10 μg in 60 μl saline) or mock-treated. Level of UQCRC2 in the airways was assessed immunohistochemically. We observed focal down-regulation of UQCRC2 (Fig. 6) in the bronchial epithelium; however, the expression of mitochondrially synthesized Cox IIb was not affected (Fig. 6). ASO-treated mice were intranasally challenged with RWE and the allergic inflammation was evaluated 72 h later (3). Differential cell counts from bronchoalveolar lavage fluid (BALF) showed that UQCRC2 antisense oligonucleotide treatment resulted in a 4.4-fold increase in the number of eosinophils compared to mock-treated, RWE-challenged mice (Fig. 7A). UQCRC2-specific ASO treatment also enhanced the accumulation of inflammatory cells in the peribronchial region of the airways (Fig. 7B). Although ASO treatment down-regulated UQCRC1 expression (Fig. 6), it did not increased significantly (P=0.08) the RWE-induced accumulation of eosinophils in the airways (Fig. 7A, B). ASO to UQCRC2 or UQCRC1 alone did not increase number of eosinophils in BALF or recruit inflammatory cells to the peribrochial area (data not shown).

FIG. 6
Inhibition of the expression of mitochondrial respiratory complex III core proteins in the lungs by local ASO treatment. ASO were administered intranasally to RWE-sensitized mice and expression of UQCRC1 and UQCRC2 was analyzed in lung sections by fluorescent ...

FIG. 7
Preexisting mitochondrial dysfunction induced by UQCRC2 downregulation increases RWE-induced accumulation of inflammatory cells in the airways. Antisense oligonucleotide treatment, specific for UQCRC2 but not for UQCRC1, increases the number of eosinophils ...
Mucous cell proliferation and hypersecretion of airway mucin are important pathological features of asthma and allergic airway inflammation (30). The levels of MUC5AC, which is the most abundant mucin produced in the airway epithelial cells during allergic inflammation (31), were determined in the BALF by ELISA. The levels of the MUC5AC were 2.4-fold higher in the BALF of RWE-challenged mice treated with ASO for UQCRC2 compared to mock-treated, RWE-challenged ones (Fig. 8A). As expected, altered expression of UQCRC2 also increased the metaplasia of mucuos cells in airway epithelium (Fig. 8B). Down-regulation of UQCRC1 did not increase significantly (P=0.09) either the MUC5AC levels in BALF or mucous cell metaplasia compared to mice RWE-challenged only (Fig. 8A, B). Antisense oligonucleotide treatment to UQCRC2 or UQCRC1 alone did not induce mucous cell proliferation and metaplasia.

FIG. 8
Mitochondrial dysfunction induced by UQCRC2 downregulation enhances RWE-induced mucin production in the airways. Down-regulation of UQCRC2, but not UQCRC1, increases the levels of MUC5AC in the bronchoalveolar lavage fluids (A) and increases the metaplasia ...
As expected, airway hyperresponsiveness was increased (as reflected by Penh values) in all RWE-challenged groups of mice compared to PBS-challenged ones (Fig. 9). However, mice treated with antisense for UQCRC2 (Penh = 62.50±9.53) but not those for UQCRC1 (Penh = 28.42±2.49), showed a significant increase (p<0.01) in airway hyperresponsiveness compared to mice challenged with RWE only (Penh = 10.85±3.17) (Fig. 9). Penh values, in mice treated with ASO to UQCRC1 or UQCRC2 alone, were similar to those in saline challenged control group (data not shown).

FIG. 9
Preexisting mitochondrial dysfunction mediated by ASO to UQCRC2 increases RWE-induced airway hyperresponsiveness. Changes in pause of breathing (Penh) as an index of airway obstruction were measured by barometric whole-body plethysmography. **P< ...
Go to:
Oxygen radical production is increased in airway inflammatory disorders, and exposure to exogenous oxidants such as cigarette smoke, diesel exhaust or ozone cause exacerbation of allergic inflammation. These exogenous oxidants also induce mitochondrial dysfunction, which is associated with the pathomechanism of inflammatory diseases. Here, we show that exposure of airway epithelial cells to RWE induces mitochondrial dysfunction via oxidative modifications of mitochondrial respiratory complex I and III proteins. Importantly, preexisting mitochondrial dysfunction induced in airway epithelium of sensitized Balb/c mice exacerbates RWE challenge-induced accumulation of eosinophils, the mucin levels and airway hyperresponsiveness. Based on these experiments we may conclude that preexisting mitochondrial dysfunction induced by oxidant environmental pollutants could be a risk factor for development of allergic disorders in atopic individuals.

In addition to mitochondrial complex subunit proteins, several accessory proteins were oxidatively damaged in the mitochondria from RWE-treated cells. The 75-kDa glucose-regulated protein (GRP75/HSP75/mortalin/TRAP-1), HSP60 and HSP70 are essential mitochondrial chaperones. Their synthesis is elevated under oxidative stress conditions suggesting a protective role against oxidative insults in mitochondria (32, 33). VDAC1 is a multifunctional protein (34) shown to interact with cytoskeletal elements (35, 36) and enzymes in the mitochondrial inner membrane (37, 38). Its damage by ROS correlates well with the observation that mitochondrial ROS are transported to the cytoplasm by voltage-dependent anion channels (39). Citrate synthase (CISY) was copurified with complex II and identified as oxidatively damaged protein in mitochondria from RWE-treated cells. Since HSP70 with its co-chaperones can bind nonnative states of proteins to prevent aggregation and assist to reach a functional conformation (40, 41), we suppose that HSP70 links carbonylated CISY and VDAC1 to complex II. Although the roles of these proteins are significant in maintaining mitochondrial integrity and function, we have focused on respiratory complex proteins, because of their direct involvement in mitochondrial ROS generation. After treatment of cultured epithelial cells with RWE, we have identified NDUFS1 and NDUFS2 from the catalytic core of the complex I, as well as UQCRC1 and UQCRC2 structural proteins from complex III, as oxidatively-damaged complex proteins. These findings were intriguing because complex I and complex III are the major sites of ROS generation in the mitochondria (42). Moreover, mitochondria isolated from RWE-treated cells showed increased release of H2O2 and suggest that oxidative damage to respiratory chain proteins are directly related to mitochondrial dysfunction. To identify the site of ROS generation in oxidatively damaged mitochondria, we used inhibitors of respiratory chain complexes. Our results show that the combined use of rotenone [inhibits the electron flow at complex I near the binding site for ubiquinol, the electron acceptor for complex I (42)] and 3-NPA [irreversibly binds to succinate dehydrogenase in the complex II (43)] blocked H2O2 generation in mitochondria from RWE-treated cells, indicating that the electron transport chain, but not alpha-ketoglutarate dehydrogenase in Krebs cycle (44), is the major source of ROS. Stigmatellin [blocks complex III at the Qo site; at sub micromolar concentrations (28)] also abolished the mitochondrial ROS generation. In contrast, antimycin A, which binds to matrix side of complex III and inhibits the Qi site of cytochrome c oxidoreductase, in the cytochrome b subunit (12), further increased the RWE-treatment induced mitochondrial ROS production. These studies together suggest that complex III is the main site for ROS generation in mitochondria of RWE-exposed cells.

Both UQCRC1 and UQCRC2 are localized to the matrix side of the complex III, in proximity to the predicted site of ROS generation at Qi center. These proteins provide structural stability to complex III. Furthermore, they have mitochondrial processing peptidase (MPP) activity, by which they process and allow proper protein folding in mitochondrial inner membrane (45). UQCRC1 and UQCRC2 have been identified among the oxidatively damaged proteins in mitochondria from kidney (46), skeletal muscle (47) and heart (47) in aging mice. Moreover, UQCRC2 was found to be involved in the natural senescence of mouse brain (48). Oxidative stress that occurs in response to pathogens also induces oxidative modification (e.g., carbonylation) to these core proteins (49). Taken together, our results suggest that core proteins in complex III are the primary acceptors of reactive radicals, and oxidative damage-induced alterations in their structure and/or function may lead to increased mitochondrial ROS production.

Protein degradation and increased replacement of modified proteins have been recognized as important factors for the maintenance of cellular homeostasis and survival after oxidative insults (50, 51). Failure or defect in the maintenance systems results in sub-physiological levels of functional proteins (accumulation of oxidatively modified and dysfunctional proteins) (29). In order to mimic the transient decrease in levels and/or functions of the oxidatively damaged UQCRC1 and UQCRC2, we down-regulated their expression by antisense oligonucleotides prior to RWE challenge. We found that down-regulation of UQCRC2, but not UQCRC1 enhanced cellular ROS levels. Although UQCRC1 and UQCRC2 share many characteristics, it seems that they have different roles in maintenance ubiquinol-cytochrome c reductase activity of complex III. Partial processing of UQCRC2 is shown to be associated with impairment of proton pumping suggesting that UQCRC2 has important role in the maintenance of inner membrane potential thereby in mitochondrial ROS generation (52). UQCRC2 is required for the assembly of complex III possessing two ubiquinone-reactive centers through which electrons are forwarded to cytochrome c oxidase. The Qo center, where ubiquinol is oxidized by redox active centers cytochrome c1 and the ‘Rieske’ [2Fe-2S] protein, is oriented toward the intermembrane space; while the Qi center, where ubiquinone is reduced by the redox center cytochrome b, is facing the matrix (53). Antimycin A inhibits Qi center thus increases H2O2 release from mitochondria. Taking this analogy, RWE-mediated oxidative damage to UQCRC2 might perturb electron flow at Qi center and thus directs electrons to intermembrane space to reduce molecular oxygen inducing elevated mitochondrial ROS production.

Mitochondrial dysfunction represents an important early step in the chain of events leading to the initiation and progression of inflammatory diseases such as diabetes, cardiovascular diseases, Parkinson's disease, Alzheimer's disease and others (54, 55). Several observations also suggest the involvement of mitochondria in allergic asthma. There is an epidemiological link between maternal history of atopy and susceptibility to atopic disorders of the descendent (56, 57). Since mitochondria are inherited through the maternal line, it raises the possibility that sequence variation in the mitochondrial genome contributes to the pathogenesis of asthma. Indeed, a mitochondrial haplogroup has been shown to be associated with total serum IgE levels in asthmatics (58). In contrast, mitochondrial haplogroups were not associated with altered lung functions or airway responsiveness (58). Mitochondrial dysfunction and ultrastructural changes (loss of cristae and swelling) have also been observed as consequences of airway inflammation in an experimental allergic asthma model (59). In the bronchial epithelium of inflamed lungs, the expression of 17-kDa protein of respiratory complex I [nuclear encoded (60)], and subunit III of cytochrome c oxidase [encoded by mitochondria (61)] was reduced (59). Our finding that in RWE-treated cells all of the carbonylated mitochondrial proteins are nuclear encoded is not a surprise, because the entire protein coding capacity of mitochondrial DNA is devoted to the synthesis of only 13 essential subunits of the respiratory complexes. Thus the majority of respiratory proteins and all of the other gene products necessary for the myriad mitochondrial functions are derived from nuclear genes (62). Based on our observations, the nuclear encoded mitochondrial proteins are more susceptible to oxidative damage than mitochondrial encoded ones; however, the presence of proteins encoded by the mitochondrial genome is also essential for elevated mitochondrial ROS production. In support, mitochondrial DNA-depleted ρ0 cells (63) did not sustain increased ROS levels after RWE exposure. Taken together, these data suggest that mitochondrial genes preferentially affect atopic diathesis (58), while defect or polymorphism of mitochondria protein encoding nuclear genes could be related to severe allergic symptoms. Further studies are needed to investigate genetic polymorphisms and distinct susceptibilities to oxidative damage of potential mitochondrial target proteins in atopic and nonatopic subjects.

In our work we show that besides the well-characterized oxidant environmental pollutants, exposure to pollen grains also trigger mitochondrial dysfunction in airway epithelial cells. Previous studies on human subjects have reported that nasal exposure to ozone (64) or particulate matter air pollution (65) followed by challenge with allergen significantly enhanced the allergic responses. Similarly, repeated pollen exposure amplified and sustained allergic symptoms (66). Whether repeated antigenic stimuli alone or together with existing mitochondrial dysfunction are responsible for this phenomenon should be investigated.

In conclusion, we have provided evidence that preexisting oxidative damage to mitochondria, prior the recruitment of inflammatory cells to the airways, intensified airway eosinophilia, increased mucin production, as well as enhanced bronchial hyperresponsiveness. Our findings further emphasize the role of mitochondria in the pathogenesis of allergic airway inflammation and asthma. Furthermore, they also imply that prevention or reduction of oxidative mitochondrial damage in the airways may have a beneficial role in therapy of these diseases.

FIG. 10
Go to:
1This work was supported by NIAID, P01 AI062885-01 (I.B., S.S.), NIH HL071163 (S.S., I.B), NIH NHLBI Proteomics Initiative, NO1HV-28184 (AK) and NIEHS Center Grant, EOS 006677 (IB, AK).

3Abbreviations used in this paper: ASO, antisense oligonucleotides; BALF, bronchoalveolar lavage fluid; Cox IIb, cytochrome c oxidase subunit IIb; DAPI, 4,6'-diamidino-2-phenylindole; DCF, dichlorofluorescein; DNPH, 4-dinitrophenylhydrazine; DPI, diphenylene iodinium; H2DCF-DA, 2′-7′-dihydro-dichlorofluorescein diacetate; NDUFS1, NADH dehydrogenase (ubiquinone) Fe-S protein 1; NDUFS2, NADH dehydrogenase (ubiquinone) Fe-S protein 2; NAC, N-acetyl-L-cysteine; 3-NPA, 3-nitropropionic acid; MRC, mitochondrial respiratory complex; ROS, reactive oxygen species; RWE, ragweed pollen extract; UQCRC1, ubiquinol-cytochrome c reductase core protein I; UQCRC2, ubiquinol-cytochrome c reductase core protein II.

Go to:
1. Bacsi A, Dharajiya N, Choudhury BK, Sur S, Boldogh I. Effect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis. J Allergy Clin Immunol. 2005;116:836–843. [PMC free article] [PubMed]
2. Bacsi A, Choudhury BK, Dharajiya N, Sur S, Boldogh I. Subpollen particles: carriers of allergenic proteins and oxidases. J Allergy Clin Immunol. 2006;118:844–850. [PMC free article] [PubMed]
3. Boldogh I, Bacsi A, Choudhury BK, Dharajiya N, Alam R, Hazra TK, Mitra S, Goldblum RM, Sur S. ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. J Clin Invest. 2005;115:2169–2179. [PMC free article] [PubMed]
4. Dharajiya N, Choudhury BK, Bacsi A, Boldogh I, Alam R, Sur S. Inhibiting pollen reduced nicotinamide adenine dinucleotide phosphate oxidase-induced signal by intrapulmonary administration of antioxidants blocks allergic airway inflammation. J Allergy Clin Immunol. 2007;119:646–653. [PMC free article] [PubMed]
5. Bowler RP. Oxidative stress in the pathogenesis of asthma. Curr Allergy Asthma Rep. 2004;4:116–122. [PubMed]
6. Riedl MA, Nel AE. Importance of oxidative stress in the pathogenesis and treatment of asthma. Curr Opin Allergy Clin Immunol. 2008;8:49–56. [PubMed]
7. Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis. 2007;12:913–922. [PubMed]
8. Servais S, Boussouar A, Molnar A, Douki T, Pequignot JM, Favier R. Age-related sensitivity to lung oxidative stress during ozone exposure. Free Radic Res. 2005;39:305–316. [PubMed]
9. Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, Wang M, Oberley T, Froines J, Nel A. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect. 2003;111:455–460. [PMC free article] [PubMed]
10. Fahn HJ, Wang LS, Kao SH, Chang SC, Huang MH, Wei YH. Smoking-associated mitochondrial DNA mutations and lipid peroxidation in human lung tissues. Am J Respir Cell Mol Biol. 1998;19:901–909. [PubMed]
11. Giles RE, Blanc H, Cann HM, Wallace DC. Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci U S A. 1980;77:6715–6719. [PMC free article] [PubMed]
12. Bacsi A, Woodberry M, Widger W, Papaconstantinou J, Mitra S, Peterson JW, Boldogh I. Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells. Mitochondrion. 2006;6:235–244. [PMC free article] [PubMed]
13. King MP, Attardi G. Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol. 1996;264:304–313. [PubMed]
14. Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, Gillespie MN. Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death. Am J Physiol Lung Cell Mol Physiol. 2002;283:L205–210. [PubMed]
15. Aguilera-Aguirre L, Gonzalez-Hernandez JC, Perez-Vazquez V, Ramirez J, Clemente-Guerrero M, Villalobos-Molina R, Saavedra-Molina A. Role of intramitochondrial nitric oxide in rat heart and kidney during hypertension. Mitochondrion. 2002;1:413–423. [PubMed]
16. Saavedra-Molina A, Uribe S, Devlin TM. Control of mitochondrial matrix calcium: studies using fluo-3 as a fluorescent calcium indicator. Biochem Biophys Res Commun. 1990;167:148–153. [PubMed]
17. Schagger H. Blue-native gels to isolate protein complexes from mitochondria. Methods Cell Biol. 2001;65:231–244. [PubMed]
18. Van Coster R, Smet J, George E, De Meirleir L, Seneca S, Van Hove J, Sebire G, Verhelst H, De Bleecker J, Van Vlem B, Verloo P, Leroy J. Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr Res. 2001;50:658–665. [PubMed]
19. Forbus J, Spratt H, Wiktorowicz J, Wu Z, Boldogh I, Denner L, Kurosky A, Brasier RC, Luxon B, Brasier AR. Functional analysis of the nuclear proteome of human A549 alveolar epithelial cells by HPLC-high resolution 2-D gel electrophoresis. Proteomics. 2006;6:2656–2672. [PubMed]
20. Trinidad JC, Specht CG, Thalhammer A, Schoepfer R, Burlingame AL. Comprehensive identification of phosphorylation sites in postsynaptic density preparations. Mol Cell Proteomics. 2006;5:914–922. [PubMed]
21. Estabrook RW, Ronald WEaMEP. Methods in Enzymology. Academic Press; 1967. Mitochondrial respiratory control and the polarographic measurement of ADP : O ratios; pp. 41–47.
22. Boldogh I, Aguilera-Aguirre L, Bacsi A, Choudhury BK, Saavedra-Molina A, Kruzel M. Colostrinin decreases hypersensitivity and allergic responses to common allergens. Int Arch Allergy Immunol. 2008;146:298–306. [PubMed]
23. Adler A, Cieslewicz G, Irvin CG. Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol. 2004;97:286–292. [PubMed]
24. Sur S, Wild JS, Choudhury BK, Sur N, Alam R, Klinman DM. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J Immunol. 1999;162:6284–6293. [PubMed]
25. Van Gestelen P, Asard H, Caubergs RJ. Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases. Plant Physiol. 1997;115:543–550. [PMC free article] [PubMed]
26. Loschen G, Azzi A, Richter C, Flohe L. Superoxide radicals as precursors of mitochondrial hydrogen peroxide. FEBS Lett. 1974;42:68–72. [PubMed]
27. Xia T, Kovochich M, Nel AE. Impairment of mitochondrial function by particulate matter (PM) and their toxic components: implications for PM-induced cardiovascular and lung disease. Front Biosci. 2007;12:1238–1246. [PubMed]
28. Degli Esposti M, Ghelli A, Crimi M, Estornell E, Fato R, Lenaz G. Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists. Biochem Biophys Res Commun. 1993;190:1090–1096. [PubMed]
29. Bulteau AL, Szweda LI, Friguet B. Mitochondrial protein oxidation and degradation in response to oxidative stress and aging. Exp Gerontol. 2006;41:653–657. [PubMed]
30. Tanizaki Y, Kitani H, Okazaki M, Mifune T, Mitsunobu F, Kimura I. Mucus hypersecretion and eosinophils in bronchoalveolar lavage fluid in adult patients with bronchial asthma. J Asthma. 1993;30:257–262. [PubMed]
31. Thornton DJ, Carlstedt I, Howard M, Devine PL, Price MR, Sheehan JK. Respiratory mucins: identification of core proteins and glycoforms. Biochem J. 1996;316(Pt 3):967–975. [PMC free article] [PubMed]
32. Mitsumoto A, Takeuchi A, Okawa K, Nakagawa Y. A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress. Free Radic Biol Med. 2002;32:22–37. [PubMed]
33. Ghosh S, Janocha AJ, Aronica MA, Swaidani S, Comhair SA, Xu W, Zheng L, Kaveti S, Kinter M, Hazen SL, Erzurum SC. Nitrotyrosine proteome survey in asthma identifies oxidative mechanism of catalase inactivation. J Immunol. 2006;176:5587–5597. [PubMed]
34. Lemasters JJ, Holmuhamedov E. Voltage-dependent anion channel (VDAC) as mitochondrial governator--thinking outside the box. Biochim Biophys Acta. 2006;1762:181–190. [PubMed]
35. Linden M, Karlsson G. Identification of porin as a binding site for MAP2. Biochem Biophys Res Commun. 1996;218:833–836. [PubMed]
36. Kusano H, Shimizu S, Koya RC, Fujita H, Kamada S, Kuzumaki N, Tsujimoto Y. Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC. Oncogene. 2000;19:4807–4814. [PubMed]
37. Juhaszova M, Wang S, Zorov DB, Nuss HB, Gleichmann M, Mattson MP, Sollott SJ. The identity and regulation of the mitochondrial permeability transition pore: where the known meets the unknown. Ann N Y Acad Sci. 2008;1123:197–212. [PubMed]
38. Roman I, Figys J, Steurs G, Zizi M. In vitro interactions between the two mitochondrial membrane proteins VDAC and cytochrome c oxidase. Biochemistry. 2005;44:13192–13201. [PubMed]
39. Han D, Antunes F, Canali R, Rettori D, Cadenas E. Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem. 2003;278:5557–5563. [PubMed]
40. Bukau B, Horwich AL. The Hsp70 and Hsp60 chaperone machines. Cell. 1998;92:351–366. [PubMed]
41. Lee GJ, Vierling E. A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein. Plant Physiol. 2000;122:189–198. [PMC free article] [PubMed]
42. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem. 2003;278:36027–36031. [PubMed]
43. Coles CJ, Edmondson DE, Singer TP. Inactivation of succinate dehydrogenase by 3-nitropropionate. J Biol Chem. 1979;254:5161–5167. [PubMed]
44. Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005;7:1140–1149. [PubMed]
45. Deng K, Shenoy SK, Tso SC, Yu L, Yu CA. Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex. J Biol Chem. 2001;276:6499–6505. [PubMed]
46. Choksi KB, Nuss JE, Boylston WH, Rabek JP, Papaconstantinou J. Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes. Free Radic Biol Med. 2007;43:1423–1438. [PMC free article] [PubMed]
47. Choksi KB, Nuss JE, Deford JH, Papaconstantinou J. Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes. Free Radic Biol Med. 2008;45:826–838. [PMC free article] [PubMed]
48. Lad SP, Yang G, Scott DA, Chao TH, Correia Jda S, de la Torre JC, Li E. Identification of MAVS splicing variants that interfere with RIGI/MAVS pathway signaling. Mol Immunol. 2008;45:2277–2287. [PubMed]
49. Wen JJ, Garg N. Oxidative modification of mitochondrial respiratory complexes in response to the stress of Trypanosoma cruzi infection. Free Radic Biol Med. 2004;37:2072–2081. [PubMed]
50. Shringarpure R, Grune T, Davies KJ. Protein oxidation and 20S proteasome-dependent proteolysis in mammalian cells. Cell Mol Life Sci. 2001;58:1442–1450. [PubMed]
51. Jung T, Grune T. The proteasome and its role in the degradation of oxidized proteins. IUBMB Life 2008
52. Cocco T, Di Paola M, Papa S, Lorusso M. Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity. Biochemistry. 1998;37:2037–2043. [PubMed]
53. Iwata S, Lee JW, Okada K, Lee JK, Iwata M, Rasmussen B, Link TA, Ramaswamy S, Jap BK. Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science. 1998;281:64–71. [PubMed]
54. Beal MF. Mitochondria take center stage in aging and neurodegeneration. Ann Neurol. 2005;58:495–505. [PubMed]
55. Mancuso C, Scapagini G, Curro D, Giuffrida Stella AM, De Marco C, Butterfield DA, Calabrese V. Mitochondrial dysfunction, free radical generation and cellular stress response in neurodegenerative disorders. Front Biosci. 2007;12:1107–1123. [PubMed]
56. Soto-Quiros ME, Silverman EK, Hanson LA, Weiss ST, Celedon JC. Maternal history, sensitization to allergens, and current wheezing, rhinitis, and eczema among children in Costa Rica. Pediatr Pulmonol. 2002;33:237–243. [PubMed]
57. Litonjua AA, V, Carey J, Burge HA, Weiss ST, Gold DR. Parental history and the risk for childhood asthma. Does mother confer more risk than father? Am J Respir Crit Care Med. 1998;158:176–181. [PubMed]
58. Raby BA, Klanderman B, Murphy A, Mazza S, Camargo CA, Jr, Silverman EK, Weiss ST. A common mitochondrial haplogroup is associated with elevated total serum IgE levels. J Allergy Clin Immunol. 2007;120:351–358. [PubMed]
59. Mabalirajan U, Dinda AK, Kumar S, Roshan R, Gupta P, Sharma SK, Ghosh B. Mitochondrial structural changes and dysfunction are associated with experimental allergic asthma. J Immunol. 2008;181:3540–3548. [PubMed]
60. Smeitink J, Loeffen J, Smeets R, Triepels R, Ruitenbeek W, Trijbels F, van den Heuvel L. Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I. Hum Genet. 1998;103:245–250. [PubMed]
61. You KR, Wen J, Lee ST, Kim DG. Cytochrome c oxidase subunit III: a molecular marker for N-(4-hydroxyphenyl)retinamise-induced oxidative stress in hepatoma cells. J Biol Chem. 2002;277:3870–3877. [PubMed]
62. Scarpulla RC. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev. 2008;88:611–638. [PubMed]
63. Chandel NS, Schumacker PT. Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS Lett. 1999;454:173–176. [PubMed]
64. Molfino NA, Wright SC, Katz I, Tarlo S, Silverman F, McClean PA, Szalai JP, Raizenne M, Slutsky AS, Zamel N. Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects. Lancet. 1991;338:199–203. [PubMed]
65. Hauser R, Rice TM, Krishna Murthy GG, Wand MP, Lewis D, Bledsoe T, Paulauskis J. The upper airway response to pollen is enhanced by exposure to combustion particulates: a pilot human experimental challenge study. Environ Health Perspect. 2003;111:472–477. [PMC free article] [PubMed]
66. Ciprandi G, Ricca V, Landi M, Passalacqua G, Bagnasco M, Canonica GW. Allergen-specific nasal challenge: response kinetics of clinical and inflammatory events to rechallenge. Int Arch Allergy Immunol. 1998;115:157–161. [PubMed]

Friday, March 15, 2013

Everything but the Kitchen Sink Quick Bread

(gluten free, corn free, dairy free, soy free, nut and seed free)

It was a clean out the fridge day. A little of this and a little of that... and presto an odd but delicious bread! Admittedly, I have been thinking about a quick bread for Abby the last few weeks. She cannot have zucchini, banana,nuts, squash. This time of year as the farmers markets start to open I start seeing terrific quick breads popping up all over the blogs. Sara said after tasting this loaf that it shouldn't but it oddly reminded her of banana nut bread. Subtle flavors with a bit of crunch and some sweet bites of figs. Just as easy as banana bread just as spicy as a zucchini- and the chickpeas were the perfect replacement for nuts. The tamarind was not noticed on it's own in the bread(not sour or tart) but did lend to add some depth.

Just a reminder, always make sure your ingredients are safe for your allergies- I post what works for Abby, which may or may not work for others with similar allergies.

preheat to 350 degrees

2 1/2 cup all purpose gf flour(posted our blend a few days ago)
1-2/3 C Sugar (domino's)
1-1/2 t (corn-free) Baking Powder(Hains)
1-1/4 t Baking Soda
1 t Salt
1 teaspoon ground ginger(we have been using mostly Simply Organic brand spices safely)
1 teaspoon allspice
1/2 cup grapeseed oil
1/2 C mashed cooked sweet potato puree(or pureed carrot? or squash? Banana? )
1 cup coconut milk(homemade or safe canned)
1 Tablespoon Braggs apple cider
2 Eggs
2-3 Tablespoons tamarind puree(I start with about 1/4 block tamarind fruit no seeds. I add a couple tablespoons hot water and stir well and then push through a fine screen until I have 2 tablespoons)
1/2 roasted chickpeas( or nuts or seeds of your choice if you can.)
1/2 cup diced dried figs(or raisins or longans or dates of your choice if you can)I soak my figs in hot water to soften.

Mix all your liquids including your sweet potato together well. Add dry ingredients and mix till just mixed - don't over beat it. Pour into 2 greased loaf pans(or 1 loaf pan and make muffins with the other half.) Bake for 60 minutes or until toothpick/knife comes out clean.

Wednesday, March 13, 2013

Chickpea Pralines

Yet another dairy free, corn free, seed and nut free, soy free, gluten free chickpea recipe!

I admit, I think we are nearly worn out on roasted chickpeas- how many can you really eat anyway? A lot.

Pralines are one of the candies that I think I most often associate with living in the South. Growing up in Seattle and NW you won't find careful wrapped pralines at the checkout line in a basket on the counter. When we moved here I found them the most satisfying 99 cent treat on my way through the line, they have been missed.

This one is easy- really. The lightly salted roasted chickpeas with the candy, is as good though different then the original.

1 cup light brown sugar(Domino's)
1/2 cup granulated sugar(Domino's)
1/2 cup fullfat coconut milk(you want to use as much of the cream and not the water as possible)
4 tablespoons Palm Shortening(Spectrum)
2 tablespoons water
1 cup roasted chickpeas

In a heavy-bottomed saucepan, combine the light brown sugar, granulated sugar, coconut milk, palm shortening, and water. Place over a medium-high heat and stir constantly until it reaches 238 to 240 degrees F. Add the chickpea's to the candy, and remove the pan from the stove. Continue to stir the candy vigorously with a wooden spoon until the candy cools, and it starts to hold it shape a bit, about 2-3 minutes. Spoon the chickpea candy out onto a parchment lined sheet pan and cool completely before serving.

Coconut Cheese.

Basically a dry coconut milk "cream cheese."

I have made yogurt cheese in the past with her coconut yogurt but it was a lot of work. This last few months I have been thinking a lot about whether it is even possible to make a coconut cheese.

The reason why dairy makes "cheese" is because of fat, protein and lactose(if you are making a hard cultured cheese, for soft cheese not needed).

Coconut milk has plenty of fat. Through making coconut yogurt I have found I can feed the bacteria by adding a bit of sugar to the coconut milk since coconut milk lacks lactose. What eluded me was the protein. Out of all the alternative milks coconut has the lowest amount of protein- really almost none.

This week I decided to try adding some rice protein. It worked. The flavor still needs work, but it melts and cooks into food like a cream cheese. When blended with eggs, nofu and plenty of sugar it made a lovely cheesecake. Eating it as is, I find it a bit gritty and too tart. I found it took quite a bit of lemon juice to get my "whey" to separate.

(I used a favorite dairy cheesecake recipe and simply substituted the cream cheese with my coconut cream cheese and nofu. I used 3/4 coconut cream cheese and 1/4 cup nofu-based on the amazing,creamy,rich results you could use all coconut cream cheese.)
Aprox 16 ounces high fat coconut milk

2 teaspoons rice protein

2-3 tablespoons fresh lemon juice.

I heated the coconut milk with rice protein mixed in until it was 190 degrees.(with the protein it will scald so heat carefully)

I cooled it to about 150. I tried adding the lemon juice around the 180 point and it did not seem to work as well. Around 150 I dripped the lemon juice across the top of the milk and allowed it to sit for about 30-45 minutes until room temperature.

Finally, I poured it into a muslin lined strainer. Cheesecloth even doubled is too porous. The coconut milk doesn't "curd" like dairy.

After about 2 hours a lot of the whey(water, should be nearly clear with a tinge of lemon but not milky at all)had drained. I tied up the top muslin like a bag and put it in the fridge to drain over night.

This morning it was still a little too soft(coconut oils harden and the water was trapped)so I simply heated it for a few in the micro and allowed to drain for another hour in muslin. Scraped it into a saran lined small pan and 2 hours later- Presto! Faux coconut cheese.

Next up I am dying to figure out if I could make a cultured cheese? It will at least be fun trying!

Monday, March 11, 2013

30 Minutes Worth of Medicine

Spent 30 minutes simply copying and pasting every medication/supplement/medical intervention that was mentioned on various Mito Blogs and groups that I have access to(I don't follow many these days.)that have been posted in the last day or so.. Buckle your seatbelt's!

Antibiotics, antibiotics for resistant infections and more antibiotics


carnitine carnitor


Digestive enzymes

Vitamin B(multi, 2,6, 12)





PICC,PEG,TPN and all the "medical" nutrition that goes with them.

EPI 743

Miralax(no studies that prove this is safe for children, mounting evidence shows this can have crazy side effects that for Abby and some others mimicked progression)


VEST(airway assistance)


Muscle biopsy, fresh,frozen




melatonin(fyi- no studies that show this is safe for under 18)







Granted, each patients don't take all of these, but some come close. In 30 minutes of simply scanning just a couple out of hundreds of pages these terms came up repeatedly.

Antibiotic resistance is up- up so high, healthy Americans are dying from resistant bacteria. Vitamins have no oversight- no idea what are in those pills. CT Scans- radiation. Recent studies indicate too many Americans, and way way too many children are taking mind alternating medication to control perceived behavioral issues in order to make caring for them easier for daycare,public school etc.

The big question, Does any of it work? Anyone recover or stop the progression with all of these interventions? I am sure some infections have been cleared up. But only to allow the next set of bacteria and fungus to cling to the lines. Anti seizure meds seem to work, but some cause other secondary issues.

Throwing spaghetti at the wall and hoping it sticks.
Copyright 2009 Abby Mito. Powered by film izle film izle favoriblog blogger themes izle harbilog jigolo