I was going to suggest that you investigate the effect of the compound on the level of expression of the genes for the various dehydrogenase enzymes known to be in the bacteria. But if the compounds are killing the bacteria, it doesn't seem sensible to do this. The apparent increased dehydrogenas activity could be an artifact of the death of the bacteria, releasing the dehydrogenase enzymes into the medium.

what kind do you use to measure dehydrogenase activity?

Dear Tomasz,

            These enzymes are the indices for chemical stress mainly tissue damage. At least in insects, when a chemical is used, the activity of these enzymes increase. I think the chemicals may cause some destructive effects on membrane of the bacteria. I suggest to search by dehydrogenases and tissue damage.

All the best,

Hey Man,

Any specyfic dehydrogenase? Or just in general?

From what I know bacteria protect themselves from harmful compounds by either active transport outside of the cell or oxidizing the threat. That would probably be the explanation for your results.

Cheers,

Tomasz

Dear all,

Bacterias (E. coli and B. cereus) were incubated in liquid media with ionic liquids, including teophylline ion. The enzyme activity was measured by adding TTC and than incubated with it. After that we used spectrophotometry method.

We were measuring dehydrogenase as general.

Do You know any nice article, which is explaining it? It would be great.

What is also interesting, that during viability tests it seams that all bacterias are dye as those dead but there is still high enzyme activity.

Thank you all

Hello,

those chemicals may have effect for the activation of this enzyme. do you know what chemicals have effect for the activation and de activation?, you may need to do this part of study if you don't have a review to explain this.

I was going to suggest that you investigate the effect of the compound on the level of expression of the genes for the various dehydrogenase enzymes known to be in the bacteria. But if the compounds are killing the bacteria, it doesn't seem sensible to do this. The apparent increased dehydrogenas activity could be an artifact of the death of the bacteria, releasing the dehydrogenase enzymes into the medium.

Hi,

Im still new in this area of research but im used to work with dehydrogenase enzyme. There are chemical that can elevate dehydrogenase enzyme. Maybe due to the metal ions (in my case Zn).

I agree with others, seems to me that this type of enzyme is like to be in citosol (maybe) and can increase activity what  could be neasured easly. You should investigate tissue damage.

Did you try if the TTC with your ionic liquid solution wihtout bacteria gave any wavelenght changes?

The dehydroganases may be reducing your theophylline ion which could give you a product that is affecting your assay

Dear Miguel,

We did a test including only TTC without bacteria and we didn't observe any changes, but it is a good suggestion with theophylline, I will try to check this out.

Thanks

Dear Tomasz,

I suspect you are speaking of NADH and succinate dehydrogenases that are usual marker activities fo stress in bacteria, am I correct?

Foreign chemicals may indeed modify the NAD+ / NADH balance and yields unexpected results. That could be a reason of what you see.

Theophylline is a methylxanthine and therefore can be oxidized through monooxygenation not easily through dehydrogenation, therefore I think theophylline interfering with dehydrogenases not likely.

On the other hand, I understand you are using TTC (water-soluble triphenyltetrazolium chloride, right?). You should rather use iodonitrotetrazolium chloride which is much better to monitor dehydrogenase global activity in microorganisms (I joined a rather old paper about that as a pdf file joined to this message).

Why's that? Because TTC is toxic to microorganisms and TTC reduction reaction is rather sluggish, therefore it is known since a long time that standard errors on TTC assays is high. Exactly what you observe. Moreover, TTC reduction is inhibited by O2 and should ideally be carried out in anaerobiosis. Do you do that ? If not, take care to the fact that different conditions of aerobiosis could highly impact the final result and introduce by doing so a high variance in the results. Again, exactly what you observe.

So, maybe you could shift to INT (iodonitrotetrazolium chloride) instead of TTC. INT has none of these problems.

I also joined a recent (2013) review on bacterial dehydrogenases and stress.

Good luck wiht your experiments,

Prof Philippe Urban

Dear professor Urban,

Thank you for you answer, it is really important for us.

I have to say, that we already knew about issue with TTC, and I think that our experiment had been planed in a way which avoid such problems (or some of them), e.g. we investigate interactions between TTC and our liquids in anaerobic conditions (in nitrogen atmosphere) but I will also do some experiments with ITC instead TTC.

Very important information is that theophilline is not able to interact with dehydrogenases.

The papers are very interesting and might help me with work on expression of some SOS genes and oxidation stress system in bacteria.

Sincerely,

Tomasz Cłapa

Dear Tomasz,

Please pay attention to the fact that if theophylline is probably not a substrate for dehydrogenase, this does not mean it is not able to interact! Theophylline could be an inhibitor of dehydrogenase.

Best regards

Philippe

Hi Tomasz,

I do appreciate comments made by all colleagues. Whenever any living system is exposed to stress (especially chemicals that inhibit/suppress electron transport), we do find a disturbance in NAD(P)H to NAD(P)+ ratio. We do find an increase in NAD(P)H levels and such an increase can inhibit glycolysis as well (thus suppress substrate level phosphorylation). Therefore, activities of dehydrogenases increase (these dehydrogenases may not be SDH). I advise you to look into activities of various dehydrogenases.

Dear professor Urban,

I have this is my mind. We generally assumed that theophylline can influence onto cells and also maybe in some ways on dehydrogenase.

Sincerely,

Tomasz

Dear Tomasz,

Dehydrogenase, which is a soluble cytosolic enzyme, present in most eukaryotic cells, releases into culture medium upon cell death due to damage of plasma membrane. The increase of the dehydrogenase activity in culture supernatant is proportional to the number of lysed cells.

In your case, it seems that the chemicals you are testing are cytotoxic to the bacteria, leading to the dehydrogenase leakage in the media compared to the media of your control that did not expose to these chemicals.

Good Luck

Hanem

First of all elaborate the chemicals you used to treat the cells and then your question can be answered suitably.

Dear Jai,

I described shortly the chemicals here:

https://www.researchgate.net/post/Does_anyone_have_experience_with_biological_research_of_Ionic_Liquids

nowwe are trying to check if there are some proteins aggregations in the cell, hope that have some answers for that today.

so we have some new data. during using Congo red - which is staining only amyloids in the cells (hopefully) we now know that ILs are influenced on the cells. we had some positive results in cells which were threaded with ILs  than in the control.

Hi Tomasz, I would join the answer of Shapiro. If your chemicals are permebealizing cells then more enzyme are available for reaction. For ex treatment of bacterial cells with CTAB allows to measure enzymes of citric acid cycle on "whole" cells suspensions

Dear Galushko,

Thank you for your help. Last week I isolated total RNA from bacteria after incubation for 24 h with those compounds. Now I planning to find some gene sequences for oxidation and heat shock and check the level of their expression.