K. Unno, T. Kishido, and M. Morioka, “Increased expression of Hsp70 for resistance to deuterium oxide in a yeast mutant cell line,” Biological and …, vol. 26, no. June, pp. 799–802, 2003.
When I started my search for papers to read, I was basically looking for something just like this. Let’s hope this leads me to some interesting finds or at least cites work that is useful to me. To the notes:
- the intro is quite interesting. they speak about using deuterated molecules for NMR (which I’ve read about before) and using microorganisms can be a way to create macromolecules in this way. having an organism that is resilient in and resistant to D2O would go a long way in this regard, hence the motivation for adapting yeast for this purpose. I don’t really care about the purposes, I just think the science is fascinating, but it helps to have some avenues of growth for other people.
- look up citation 8
- “An inhibitory effect of D2O seems to be due to inhibition of tubulin polymerization and effects on microtubule-organizing centers and other structures governing the formation of the mitotic spindle. 9—11” – This is right in line with our research involving kinesin. Could be useful for Nadia and (former grad student now resident bad ass at UT Austin) Andy.
- The rest of that paragraph seems pretty important for sources as well: “Other effects of D2O on energy production, such as lowered ATP/ADP ratios (12), and on membrane receptors, such as impaired Ca2+ channels and Na+ -K+ AT-Pase, have also been reported (13—15). D2O also affects heat- sensitivity (16—18) and the longevity of singlet oxygen (19—21). These effects suggest that cultivation in D2O is stressful for cells. The expression and induction of molecular chaperones (heat shock protein, Hsp) might be important for growth in D2O.”
- they used the cell line ssa1ssa2 – strain A1630. I’ll have to figure out what that is and where they got it from and how that got produced. “cells were kindly donated by Dr. S. Lindquist of the University of Chicago.” Maybe Dr. Linquist can kindly donate some cells to me as well? I’m a little bummed that this paper cheated and used premutated cells.
- They’re cell cultivation isn’t all too different from mine: place cells in liquid media and stick in incubator.
- Going back through the intro, they choose this cell line because increased Hsp70 production leads to enhanced stress resistances. They make it seem like they were hoping that D2O resistance was one of the stress tolerances, and they find out it was.
- this paper is classic scientist needs to use big and exclusive words to validate his claims. ALL THAT DOES IS MAKE IT TAKE LONGER FOR PEOPLE TO READ YOUR PAPER!
- I understand the role of Hsp70 in relation to this project now: Hsp70 is a heat shock trascriptional factor inhibitor. So this cell line doesn’t produce Hsp70 which prevents the transcription factor from deactiving and thus makes lots of heat shock proteins.
- after reading a little about yeast log-phase growth I wonder if my cell growth measurements are based on the dormancy phase of growth and i’m comparing that to mid-log phase growth. hmmm…
- they do 2-d electrophoresis! still don’t know too much about their methods here though…
- so it seems from the strain ss1ss2, they compare the growth of a bunch of clones and pick two (named S-10 and S-11) whose doubling time matches the wild type strain in H2O.
- from here they discovered that the amount of Hsp70 was higher in S-11 than in the wild type (wt). which is interesting because they wanted to have as little Hsp70 as possible based on the introduction. But they then state that the expression of Hsp70 (induction is the word they use) might provide D2O resistance.
- Their data shows that these S-11 cells grow as well as the wt and the ssa1ssa2 strain (still having a hard time understanding the difference between this line and the S-11 line) in H2O, but in D2O only the S-11 line exhibits growth.
- and in H2O the amounts of Hsp70 in S-11 were several times higher than the wt, but in D2O it was only slightly more.
- The grammar of these sentences confuse me: “As the level of Hsp70s was changed among ssa1ssa2 clones(24), the clone cells with low level of Hsp70s were used. However, the increased level of Hsp70s was not observed in D2O. An increased level of Hsp70, especially one previously induced, might be important for cell growth in D2O.” It seems like a contradictory statement. They state earlier in the paper that the clone they chose over-expressed Hsp70, “We isolated a clone named S-11 that grew as fast as the wt and over-expressed Hsp70.” Which is directly opposite what they say in the first quoted text above. Then they say that Hsp70 level increases were not observed in D2O, while saying “Cultivation in D2O slightly increased the level of Hsp70 in S-11… The induction of Hsp70 was significantly found in the wt and S-10 cells.” Am I looking at the wrong things? Is this paper just very poorly written? If anyone wants to read this paper, I’d be happy to share it with them via dropbox or Mendely.
- I suppose the takeaway from this is that Hsp70 production leads to D2O resistance.
- “As a next step for obtaining highly deuterated compounds, it might be needed to culture cells using D-labeled dextrose and amino acids.” I find this interesting. I suppose I’ve taken for granted the fact that mixing YPD with 99.9% D2O doesn’t automatically mean that my peptone, and dextrose will be mostly Deuterium. I was hoping that the amount of deuterium in solution would force d-exchange toward replacing most hydrogen on the non-water molecules. Hmm. I know I can get deuterated dextrose, but can I get deuterated peptone? Should I bother?
- Citation 24 seems pretty important to read, and may shed some light on issues that confuse me.
- Unno K., Kishido T., Okada S., Biol. Pharm. Bull., 21, 631—633 (1998).
The results of this experiment seem to indicate the same thing that yesterday’s paper indicated: D2O adaptation is more a tolerance than an adoption of deuterium. Organisms that tolerate deuterium may not incorporate deuterium in vital molecules. I think this because both papers reveal that D2O adapted organisms grow normally in the presence of H2O. The difference between the two is that this paper doesn’t experiment in water after testing for D2O resistance. The yeast here is just kinda good to grow in D2O but also grows in H2O.