Mitochondrial quality regulation question

[Bioperson]

I'm wondering about the mechanisms behind the decrease of mtROS (mitochondrial reactive oxygen species) by uncoupling of oxidative phosphorylation? Can someone please explain? Thank you!

[deleted-143835]

Hi!

I assume mtROS by uncoupling of oxidative phosphorylation is the topic of your research project - what exactly are you trying to test? Do you have a hypothesis/variables mapped out yet? Knowing those may assist me in giving you a better quality answer.
In the meantime, here are some papers that might help you:
http://www.ncbi.nlm.nih.gov/pmc/article ... p00163.pdf
http://www.ncbi.nlm.nih.gov/pubmed/10075080
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1475474/

Sorry this isn't of too much help! Details would definitely help a ton!

Good luck!

[Mirza]

Hello and great question! I would be interested to know how you came up with it, because uncoupling oxidative phosphorylation (or any distruption to aerobic metabolism) will actually increase reactive oxygen species. If you disrupt the electron transport chain, eukaryotic cells have to depend on anaerobic metabolism (namely glycolysis) to meet energy demands. The cell will quickly exceed its electron carrying capacity because glycolysis produces NADH from NAD. When the cells run out of NAD they wastefully use the NADH to turn the pyruvate into lactate. This is extremely wasteful but is a desperate last attemp to keep glycolysis going with fresh NAD. The metabolic demands of a cell which relies on glycolysis alone will then create an abundance of reactive oxygen species as high energy electrons are being expelled in more destructive ways (in the process of removing those electrons reactive oxygen species must be produced). This is famously observed in avascular regions of tumors, where the cells recieve no fresh oxygen so they just put glycolysis into overdrive (known as the Warburg Effect). In the process they create many more ROS resulting in greater genetic instability associated with cancer. Hope this helps.

[Bioperson]

Hi,

Thank you very much for the replies! I really appreciate your help.

I've been doing a lot of research lately and my project has evolved a bit. The goal hasn't changed; it is still finding a relatively easy and effective method for humans to be able to regulate (or control, not necessarily meaning surpressing) mitochondria superoxide production. I think I should approach regulation of mtROS generation by altering the forces that regulate the reverse electron transport (RET) in complex I of the mitochondria; complex I has been proven to generate the largest amount of superoxide in the mitochondria by RET. My current options for attempting regulation are:
1. Using hormones (simply an idea, I still need to continue to do more research on this), since it has been proven to have significant effects in mitochondrial energetics (the paper I am now reading is called "Thyroid Hormone Effects on Mitochondrial Energetics")
2. Alter uncoupling proteins (2 and 3?) to modulate mitochondrial O2•- production
3. Alter ∆p - specifically ∆pH (the pH gradient), which super oxide production by complex is very sensitive to, even more so than to the membrane potential component of ∆p
4. Use ionophores or uncouplers to alter the components of ∆p
5. If cannot alter ∆p, alter semiquinones and semiquinolates that are formed during proton pumping that react directly with o2 to form O2•- to control superoxide production

What are your thought on my ideas?

One more concern of mine is isolation of the mitochondria, which I will have to perform in order to perform my experiment properly. Is there a protocol for isolation of the mitochondria that someone can possibly send me? That would be much appreciated! I'm also wondering if it is possible to isolate mitochondria from a cultured cell line instead of from cells fresh out of an animal?

Thanks for all help!

[deleted-140482]

Hi,

It sounds like you've done a lot of research to prepare for your project, which is great! I'm not that familiar with the ideas you are considering, so I won't comment on that part. Hopefully another expert with more expertise in the area can help you there. There are protocols to isolate mitochondria, either from animal tissue or cell culture cells. I'll attach a paper in my next post that has a protocol for isolating mitochondria from skeletal muscle which is a fairly common method. Alternatively, there are a number of kits that can be used to isolate mitochondria from cell culture. They cost about $200, so they may be outside of your price range.
http://www.piercenet.com/product/mitoch ... ured-cells
http://www.abcam.com/mitochondria-isola ... 10170.html
I'm sure you should be able to find a non-kit method as well. I'll attach one I found, but I it's just a protocol a lab in Utah uses, and since I've never used it personally, I can't vouch for its effectiveness.

What method you chose will obviously also depend on the tools available to you. You will probably need a university-level lab to do cell culture techniques. Do you have that available to you?

Hope this helps!
JMP

[deleted-140482]

Skeletal muscle isolation

[Bioperson]

Hello JMP,

I really appreciate your response. This field that I'm currently working in is not a very common one. Thus, I'm trying to bring more attention to this with my project. In response to your question, yes, I do have a lab available to me. I've worked there for my last project and thus have decent cell culture experience and over the summer with a graduate student as well.

[Bioperson]

What are some factors that regulate glycolysis within our system?

[deleted-143835]

Hi!

I did a Google search and found this link, which might help: http://cmgm.stanford.edu/biochem200/regulation/

Regulation of glycolysis, as I presume is with any metabolic pathway, depends on availability of substrate, concentration of enzymes, etc. - more details are explained in the link. Please post back if you're confused on anything!

[Bioperson]

Thanks for the link; it's really a great one. I have another question: is there a difference between the electric charge of a cancer cell and a normal cell?

[Bioperson]

actually, more specifically, is there a difference between the electric charges of the cancerous mitochondria and the normal cell's mitochondria?

[deleted-140482]

The mitochondrial membrane potential is different between normal and cancerous cells (due to the Warburg effect that Mirza mentioned), but that is not exactly the same thing as there being a charge difference in the membranes. I couldn't find any conclusive evidence that the actual charge of the membrane (i.e. how negatively or positively charged it is) changes between cancerous and non-cancerous cells, but the fact that cancerous cells have a higher membrane potential (i.e. more voltage) than normal cells is fairly well accepted although all of the reasons behind it are not well understood.

I hope that helps/answers your question.

[deleted-143835]

I think JMP gave you excellent advice. While I too can't find any conclusive evidence about the membrane charge, this link discusses the difference in cellular potentials of normal vs. cancer cells: http://cancerres.aacrjournals.org/conte ... 0.full.pdf
Hope this helps! Please post back with any more questions.

[Bioperson]

Thanks! And yes I do have another question regarding nanoparticles as drug vehicles: which kinds of nanoparticles are ideal for drug delivery into the mitochondria? I also need it to be positively charged. Also, what is the procedure for 'programming' the nanoparticle to release its drug contents at a designated spot in the cell?

[Bioperson]

Also, how can a nanoparticle be delivered through multiple biological barriers without releasing the drug content? WIth which type of nanoparticles is that possible?

[deleted-140078]

To answer the second part of your question, I would imagine it would be related to the chemical environment that is present in the interior of your target space. Solubility and pH differences can be used to construct a way to ensure most of your chemical gets released in the appropriate area. There may be a way to take advantage of any enzymes that are present as well. But using this as a key to unlock your package may require significant computer modeling and outside resources. As far an more specialized answers, I am afraid this is out of my reach. I would suggest trying to contact a University that has a nano-tech research program and explain your project, they may be able to offer some ideas.

[Bioperson]

Hi,

I am currently in need of a chemistry computer program that can help me prove the effectiveness of my nanoparticle by theory (basically able to recognize the chemical reactions between polymer and biological chemical, e.g. H2O2). Also, I'm in need of another computer program that can create 3D models that can act as a display; or even better, it can display a certain chemical reaction in a video type display.

Thank you for your help