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Prometheus vs. SENS


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#91 caston

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Posted 05 September 2006 - 01:13 PM

thanks prometheus,

I'll keep asking simple questions and try to use my imagination to help push our science along.

#92

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Posted 05 September 2006 - 01:37 PM

I'm ignorant of the many uses of RNA.

Nevertheless, your rate of knowledge absorption and ability to draw sensible conclusions in the life sciences for a non-life sciences graduate is impressive.

The functional diversity of RNA is extensive and may not be completely elucidated. In basic biology we are taught about mRNA which is read by RNA polymerase to translate into an amino acid sequence but there are so many other cell functions associated with RNA which include numerous methods of gene splicing, silencing and enzymatic activity. In some cases certain cells only have mRNA rather than DNA and a nucleus - such as red blood cells - so in such cells RNA damage would have dire consequences. In a nucleated cell, if the RNA that is damaged is of the silencing variety then genetic dysregulation can occur such as the expression of an oncogene; if associated with splicing it can once again result in innapropriate gene expression and similarly when RNA acts as an enzyme, damage would result in decreased activity. The consequences are extremely varied in scope and I must say have not been studied as much as I would have thought (a pubmed search on "RNA damage" yields only 29 results whereas a search on "DNA damage" returns over 37,000). Consequently, RNA damage could account for an enormous and unsubstantiated impact in cellular function and the various theories of aging.

#93

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Posted 05 September 2006 - 01:38 PM

thanks prometheus,

I'll keep asking simple questions and try to use my imagination to help push our science along.

Thank you, Caston. I think you've opened a Pandora's box. :)

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#94 caston

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Posted 05 September 2006 - 03:31 PM

Quick close it again!!! :)

#95 jaydfox

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Posted 05 September 2006 - 07:31 PM

I understand that damaged RNA can have potentially dire consequences for cell health, and now that you've brought up the silencing role, I can even appreciate the cancer role, sort of.

But I'm still wondering what the role of RNA damage is in aging. RNA is a short-lived molecule, at least compared to the lifetime of mammals (and especially humans). It gets used (perhaps even several times?), then at some point gets recycled. New RNA is transcribed.

SENS targets (and, to a degree, anti-aging targets in general) are/should be things which accumulate. nDNA damage accumulates because nDNA is not recylced and replaced with fresh copies. (Well, it is in mitotic tissues, where old cells are ablated. But even then, the ultimate stem cells can accumulate nDNA damage.) Lipofuscin accumulates because it cannot be broken down. Cross-linked proteins accumulate because those particular proteins are some of the few designed to last a lifetime. Extracellular protein aggregates accumulate for similar reasons to lipofuscin.

mtDNA is trickier, because there is mitochondrial autophagy which in theory should be able to eliminate poor quality mtDNA. But even then, if all mitochondria in a given period acquire at least one new mutation (actually, to be more precise, if more than half acquire a mutation in every mitochondrial turnover cycle), then autophagy cannot prevent accumulation. At best, it can make sure that only the most insignificant mutations accumulate, but over a lifetime, that could be significant. Also, some mutations may fool the autophagy system, via de Grey's SOS hypothesis (or perhaps other, similar mechanisms). But ultimately, the point is, mtDNA mutations do accumulate, at least in a fraction of cells significant enough to matter (possibly).

Does damaged RNA accumulate? If not, that doesn't mean it can't be a SENS target. But we must define an aspect of RNA damage that increases with age, and then determine if fixing the other strands will not already address the problem.

For example, if RNA damage is due to transcription of a damaged nDNA sequence, then the problem isn't really RNA damage. (Which isn't to say that repairing the RNA might not be a valid alternative to repairing the original nDNA template (though I doubt it), but that's not quite the same as saying that damaged RNA is the problem.)

Or, the problem might be an increased level of ROS that damages RNA molecules more during their relatively short lifetime. That increased ROS level could be traced back to a different SENS target, most likely mtDNA, though possibly lowered ROS-scavenging enzyme levels (or missing enzymes) due to nDNA (epi-)mutations.

Or, the problem might be an increased lifespan of RNA molecules due to reduced RNA scavenging and recycling, due perhaps to lower enzyme levels (due to nDNA (epi-)mutations), or due to damaged enzymes (due to ROS, due to mtDNA damage).

Or, the problem might be that a damaged RNA molecule somehow leads to a dynamic perpetuation of itself, by some means that I couldn't begin to contemplate because I'm ignorant of cellular biology. This latter case, a dynamic perpetuation of RNA damage, is the only one of the four examples I gave that presents itself as a valid SENS target. There are others we could come up with, but my point is, if there is damaged RNA, and if it's a problem, the problem likely traces back to an existing SENS strand (by SENS, I'm making implicit the recognition of nDNA damage as a valid SENS target, even if there isn't a proposal to fix it).

So I'm curious if there's any evidence to even lead us to believe that RNA damage accumulates in its own right. (On a sidenote, a similar line of reasoning to the above (minus the references to SENS) might be one reason that RNA damage has received so little attention.)

#96 eternaltraveler

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Posted 05 September 2006 - 07:50 PM

i don't see how RNA damage could be a particular issue. The machinery used to proof read RNA is much worse than the machinery used to proof read DNA precisely because there is a high rate of turnover of RNA, and it's non-perpetuating. If there are a few bad RNAs for any given protien, there are hundreds or thousands of good RNAs for the same protien.

Of course if the damage is upstream of the RNA (in the DNA) then all copies will be bad.

#97

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Posted 06 September 2006 - 02:33 AM

i don't see how RNA damage could be a particular issue.  The machinery used to proof read RNA is much worse than the machinery used to proof read DNA precisely because there is a high rate of turnover of RNA, and it's non-perpetuating.  If there are a few bad RNAs for any given protien, there are hundreds or thousands of good RNAs for the same protien.

Of course if the damage is upstream of the RNA (in the DNA) then all copies will be bad.


In order to evaluate the consequences of this undereported and underappreciated type of damage (of which I am as guilty as anyone up until it was pointed out by Caston), one must a) survey the extent of RNA functional diversity, and b) the turnover rate of critical RNA molecules.

Note the following abstract:

Eur J Clin Invest. 2004 May;34(5):323-7.
Reactive oxygen species induce RNA damage in human atherosclerosis.
Martinet W, de Meyer GR, Herman AG, Kockx MM.
University of Antwerp, Wilrijk, and General Hospital Middelheim, Antwerp, Belgium. wim.martinet@ua.ac.be

BACKGROUND: Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. MATERIALS AND METHODS: The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. RESULTS: Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. CONCLUSION: Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.


In this case only the the 18S and 28S ribose RNA (components associated with RNA polymerase) were studied in cells derived from arthetotic arteries. The same damage that resulted in a significant reduction of rRNA detection could also have affected other non coding RNA species including snoRNA (splicing), snRNA (splicing), miRNA (silencing), tRNA (translation), telomerase RNA (telomere maintenance), etc.

#98

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Posted 06 September 2006 - 02:36 AM

Does damaged RNA accumulate?

Interesting question. It could, if damage-mediated conformational changes resulted in its inability to be recycled.

Arch Biochem Biophys. 1997 Aug 1;344(1):200-7.
Cisplatin inhibits protein synthesis in rabbit reticulocyte lysate by causing an arrest in elongation.
Heminger KA, Hartson SD, Rogers J, Matts RL.
Department of Biochemistry and Molecular Biology, and Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater 74078-3035, USA.

The mechanism through which cisplatin (cis-diamminedichloroplatinum) inhibits protein synthesis in rabbit reticulocyte lysate was characterized. Cisplatin and transplatin caused a progressive slowing in the rate of protein synthesis culminating in the complete arrest of translation. Inhibition was dependent upon the aquation of the compounds. Addition of eukaryotic initiation factor eIF-2, eIF-2B, cAMP, MgGTP, or dithiothreitol neither prevented nor reversed the inhibition induced by cisplatin, indicating that the mechanism of cisplatin-induced translational inhibition is distinct from the inhibition induced by other toxic heavy metal ions (Hurst, R., Schatz, J. R., and Matts, R. L. (1987) J. Biol. Chem. 262, 15939-15945; Matts, R. L., Schatz, J. R., Hurst, R., and Kagen, R. (1991) J. Biol. Chem. 266, 12695-12702). Analysis of the polyribosome profile of cisplatin-inhibited reticulocyte lysate indicated that cisplatin arrests the elongation stage of protein synthesis. Agarose gel electrophoresis and Northern blot analysis indicated that mRNA and rRNA become crosslinked to form very high-molecular-weight adducts upon extraction of the RNA from polyribosomes of cisplatin-treated lysates. Diethyldithiocarbamate, which reduces the cytotoxicity of cisplatin in vivo, protects protein synthesis in reticulocyte lysate from inhibition by cisplatin. The data suggest that extensive derivatization of reticulocyte lysate RNA by cis- and transplatin results in the arrest of translating ribosomes. Since arrest of translational elongation is a well-defined mechanism of action of several families of toxins, we suggest that it may contribute to the cytotoxic action of cisplatin observed in certain populations of cells.


and,

Biochemistry. 2001 Aug 21;40(33):9977-82.
Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein.
Hayakawa H, Kuwano M, Sekiguchi M.
Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. hiroshi@med.kyushu-u.ac.jp

8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.



#99 telomolecular

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Posted 22 December 2006 - 11:51 PM

I think that there are allot of useful concepts in SENS, but until the WILT concept is eliminated from the platform it will always be an entirely philosphical paradigm with no real world use. A better tactic is to simply eliminate cancer and extend telomeres, this will result in improved cellular stability, proper cell function, and is completely doable (and in fact our group has already done it). This results in very young and healthy tissues in dividing cell types. The combination of our Notch 1 therapy regenerates musculate, neuronal tissue, and heart tissue. While stem cell therapy may represent a nice complementary technology, there is no evidence that stem cell therapies can reconstitute tissues in a normal ongoing way and it does amost nothing for aged tissues that will in one way or another degrade and corrupt as telomeres shorten. The exact differentiation of cells is important and stem cell therapy is a rather unregulated growth of new tissues in old systems. WILT can never happen in presently living creatures and would cause all sorts of undesirable side effects. I think the whole SENS concept would benefit from rethinking its stance on telomeres and incorporating their correction into the model. Otherwise its a theory that is bound to become slowly discredited and will not echo properly as planned.
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#100

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Posted 23 December 2006 - 12:11 AM

The combination of our Notch 1 therapy regenerates musculate, neuronal tissue, and heart tissue. While stem cell therapy may represent a nice complementary technology, there is no evidence that stem cell therapies can reconstitute tissues in a normal ongoing way and it does amost nothing for aged tissues that will in one way or another degrade and corrupt as telomeres shorten. The exact differentiation of cells is important and stem cell therapy is a rather unregulated growth of new tissues in old systems.

It has been a long standing concern of mine that merely injecting fresh/youthful stem cells will not work (as the literature suggests) since their phenotype changes due to the signaling they aquire from the host environment. Rather, I have considered methods of modulating their response by pretreatment with factors that would inhibit such signaling long enough for them to engraft. The alternative is your strategy which is along the lines of Conboy's work and patent. I suspect an optimal solution would employ both strategies.

#101 caston

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Posted 23 December 2006 - 06:55 AM

i don't see how RNA damage could be a particular issue.  The machinery used to proof read RNA is much worse than the machinery used to proof read DNA precisely because there is a high rate of turnover of RNA, and it's non-perpetuating.  If there are a few bad RNAs for any given protien, there are hundreds or thousands of good RNAs for the same protien.

Of course if the damage is upstream of the RNA (in the DNA) then all copies will be bad.


Just an idea but if the damaged RNA or siRNA blocks messages to DNA repair genes then we do have more upstream damage.

Edited by caston, 23 December 2006 - 07:49 AM.


#102 jaydfox

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Posted 23 December 2006 - 08:40 AM

Just an idea but if the damaged RNA or siRNA blocks messages to DNA repair genes then we do have more upstream damage.

True, but the RNA is transient while the DNA is permanent, so the repair would need to be in the DNA, as long as the damaged RNA turns over. If the damaged DNA leads to damaged RNA that perpetuates the cycle, then the RNA is not technically the problem, though interfering with it can break the cycle. If the RNA is not being degraded, but remains with a very long lifetime within the cell, then it could be considered accumulating damage of the type SENS seeks to remove.

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#103 caston

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Posted 24 December 2006 - 03:53 PM

Jay,

I think we still have a lot to learn about RNA and we will be dealing with it a lot in molecular therapy. We can take the extreme of suggesting that RNA damage is the root cause of all ageing or the other extreme of saying that it makes no difference to it at all but the reality will likely be somewhere in between.
I think you are taking the middle ground from reading your post but we still have a lot to learn about exactly how it effects things like gene expression and if damaged RNA leads to poor DNA repair.

Study up for there will be many heated debates in the future. ;)

My view has changed a lot since posting that I now think *NA repair is regulated according to what has been shown to give the best chance of survival for the mitochondria.

Edited by caston, 28 May 2007 - 01:57 AM.





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