aftereleven Posted March 28, 2008 Share Posted March 28, 2008 I'm testing the toxic effects of a protein on a certain cell line but my results dont make any sense to me. Some background info: The protein is a blue copper redox protein native to some bacteria, involved in their electron transport. it is a redox protein, and is known to interact with cell signalling pathways through transcription factors. i did an assay measuring the cell cycle or S phase activity on the cell lines using different concentrations of the protein. In this assay, higher readings indicate higher cell cycle activity. I'm using a control with no protein where the readings from it would indicate normal cell cycle activity My results are strange. Using low concentrations of the protein, the readings are lower than the control, indicating inhibition of the cell cycle, but at high concentrations of the protein i get values significantly higher than the control indicating stimulation. So my question is how can different concentrations of the same protein give two completely different outcomes? can lower concentrations stimulate different pathways than higher concentrations? I'm really stuck on this and would really appreciate any insights, thanks Link to comment Share on other sites More sharing options...
YT2095 Posted March 28, 2008 Share Posted March 28, 2008 yes, certainly in Bio systems, there is an Optimum level for Most compound to be present. it will show in many systems like a `J` curve. not enough of Chemical X and the system doesn`t perform and too much and the same happens. the J curve where the Y axis is mortality, and X axis is Dose (or concentration), the Zero line is the Average mortality/survival. Link to comment Share on other sites More sharing options...
CaptainPanic Posted March 28, 2008 Share Posted March 28, 2008 First of all, I am no expert... and I don't know much about the effects of this particular protein on bacteria. I've never heard of it. All I can do to help is give another example where a changing concentration changes the metabolic pathway: Yeast in low glucose concentrations, with oxygen present will simply grow and produce only CO2 and water (and lots of new yeast cells). If concentrations of glucose go up a lot, it switches to anaerobic growth, even with plenty of oxygen present. Growth almost completely stops, and ethanol production starts. This is well documented, and it is called the "Crabtree effect". Wikipedia has a small article about it, but I'm sure that if you're interested, more info is available: http://en.wikipedia.org/wiki/Crabtree_effect [edit]...And in addition to what YT2095 says: think of elements that are needed in spore-concentrations (ppm or lower)... many (heavy) metals are vital for life, but toxic in high concentrations. Link to comment Share on other sites More sharing options...
aftereleven Posted March 28, 2008 Author Share Posted March 28, 2008 Thanks for the replies. sorry Capt i wasnt clear, the protein is just derivied from bacteria, im actually testing it on human cells. the yeast analogy is good but i dont think i can compare the two. im testing it on tissue cells and not organisms and the protein is completely foreign to the cells and has been known to be generally toxic but little research is done on it. YT2095, the J curve seems to be what is happening with my results but i can't put a theory behind the cause I'm thinking in the context of this particular assay, i.e measuring the cell cycle. i wanna know how is it possible that a protein (or any other factor) can inhibit or appear to be inhibbiting the cycle at low concentrations but stimulate or appear to be stimulating it at higher concentrations. the only explanation i can think of is that the protein is generally toxic but at higher concentrations perhaps some defense mechanism of the cell becomes activated to counteract the effects which makes it appear like the protein is having possitive affects, but that doesnt seem likely. Link to comment Share on other sites More sharing options...
YT2095 Posted March 28, 2008 Share Posted March 28, 2008 the only explanation i can think of is that the protein is generally toxic but at higher concentrations perhaps some defense mechanism of the cell becomes activated to counteract the effects which makes it appear like the protein is having possitive affects, but that doesnt seem likely. that sounds like Classic Hormesis J curve behviour, I could be wrong as Biology is not my area, but have a look anyway, it may help provide further info as to what it Isn`t: http://en.wikipedia.org/wiki/Hormesis Link to comment Share on other sites More sharing options...
aftereleven Posted March 28, 2008 Author Share Posted March 28, 2008 hmm just had a look at that was pretty interesting. Problem is Hormesis is the exact opposite of what is happening. Hormesis seems to mean benefitial effects of toxins at low doses and the opposite at higher ones. My result suggest the opposite. Anyway thanks for the insights Link to comment Share on other sites More sharing options...
CharonY Posted March 28, 2008 Share Posted March 28, 2008 Well, basically it is not uncommon to have concentration dependent effects. It is kind of strange that a high amount has seemingly beneficial effects, though. In general, concentration-dependent effects can be unspecific. E.g. w/o knowing details one can speculate that the redox enzymes decompose and increase the amount of copper in the medium. However, I'd not assume that this would be beneficial (an iron enzyme might make more sense, depending on the cell culture). One question would be how this bacterial proteins is supposed to affect the growth of the cell line in this particular experiment? Link to comment Share on other sites More sharing options...
aftereleven Posted March 28, 2008 Author Share Posted March 28, 2008 In general, concentration-dependent effects can be unspecific. Yes i really think something non specific to the protein might be causing these changes, like certain cell repair mechanisms or other cell signalling pathways that come into effect during certain concentrations, but i dont know much about this so i cant think of any reason and have been unable to find one on the net/books that can explain the opposing changes in this context. One question would be how this bacterial proteins is supposed to affect the growth of the cell line in this particular experiment? Well from the little research done on it so far, it is supposed to be generally cytotoxic to the cell line. but the research is really small, probably like one experiment, so i'm willing to speculate any outcomes, problem is i dont see any outcome that i can explain logically Link to comment Share on other sites More sharing options...
CharonY Posted March 29, 2008 Share Posted March 29, 2008 Before you proceed thinking in that direction have you double checked that your results are statistically relevant? Moreover are the results repeatable? Was everything standardized? That is, where the experiments done in one go, or on different days? Where the cells from a single split culture or could they have different histories? Whenever something counterintuitive pops up it makes sense to be really really sure that the effects were not due to handling, different batches of cells etc. Otherwise you are going to lose a bunch of time. Link to comment Share on other sites More sharing options...
aftereleven Posted March 29, 2008 Author Share Posted March 29, 2008 well for every concentration tested i made four samples, all with the same concentrations, same cells and tested them simultaneously. i averaged them to get a single result. the error bars on my graphs seem pretty tight. while certain assays were done on different days, the different concentrations used for an assay were all tested together. besides, i have no more lab time left to repeat them, just have to stick with what i have and hope for the best. Link to comment Share on other sites More sharing options...
CharonY Posted March 30, 2008 Share Posted March 30, 2008 the different concentrations used for an assay were all tested together. So do you mean that you made a run with various concentrations on a single day and then repeated the whole assay on another (i.e. there is not likely a bias towards specific concentrations?). Regarding the analysis, "the error bars are pretty tight" is at best just a qualitative assessment. At the very least do a t-test. Link to comment Share on other sites More sharing options...
aftereleven Posted March 30, 2008 Author Share Posted March 30, 2008 So do you mean that you made a run with various concentrations on a single day and then repeated the whole assay on another (i.e. there is not likely a bias towards specific concentrations?). yes, pretty much I'm not at all ruling out the possibility that some results are wrong, im just saying i tried to conduct the experiment as accurately as i could and the stats didnt reveal any major problems. Judging by the results, something may well have gone wrong but as i said i dont have anymore lab time to repeat anything, otherwise i most likely would have with all the confusion its causing. all i can do is discuss what i have. i've come up with a few speculations that admittedly sound a bit silly, but i'd rather not simply report that i couldnt discuss anything because my results are likely wrong. Link to comment Share on other sites More sharing options...
CharonY Posted March 31, 2008 Share Posted March 31, 2008 Yes, that's why it is important to include statistics Link to comment Share on other sites More sharing options...
Recommended Posts
Create an account or sign in to comment
You need to be a member in order to leave a comment
Create an account
Sign up for a new account in our community. It's easy!
Register a new accountSign in
Already have an account? Sign in here.
Sign In Now