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 Everything
(~} ;-}
 
Registered: 06/26/10
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 Tryptamines in substrate with DPT? 
#22216074 - 09/10/15 09:23 AM (8 years, 7 months ago) |
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Basically, i am going to try this. or if a friend wants to i will give him the DPT to add to substrate.
Not even shulgin has info on dosage of 4-ho-DPT but i have a pretty good guess its around 25mg for a moderate to strong dose 
This is a project in the distant future, nothing of now. but will report back if i have results. there is seriously no info on 4-HO-DPT because shulgin says it is so hard to make but i think he meant synthetically not through the mushroom, at the time of pihkal this method was not known.
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 bodhisatta  
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22230558 - 09/13/15 09:07 AM (8 years, 7 months ago) |
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If you put it in the substrate the mycelium will just enzymaticly degrade it
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Coincidentiaoppositorum
deep psychedelic
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Re: Tryptamines in substrate with DPT? [Re: bodhisatta]
#22230585 - 09/13/15 09:14 AM (8 years, 7 months ago) |
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You are refering to jochen gartz "biotransformation of tryptamine compounds using stropharia cubensis, no?
He holds a patent on the process, he claims the enzymes do not care what tryptamine you provide them with, that they will 4-hydroxylate then 4-phosphoralyze said tryptamine, gartz claims that you need only to introduce the tryptamine to the substrate and that the 4-substituted version will be present in the cataphores.
He claims to have produced 4-ho-DET this way, and maybe another compound, I cant really remember.
Though I'm not sure if anybody has successfully reproduced these experiments.
-E. Borodin
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Everything
(~} ;-}
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thanks dude, so has anyone on the shroomery attempted this unsuccessfully?
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22234693 - 09/13/15 10:09 PM (8 years, 7 months ago) |
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Ive looked around and heard of some failures, (they said it just made the mushrooms smaller) there were also anecdotes of success, but nothing more than anecdotes.
If you do successfully reproduce gartz experiments, please share, ive been I interested in compounds like 5-methoxy-4-hydroxy-N,N-dimethyltryptamine, which are difficult to produce in the lab, but could simply be produced by gartz proposed methods.
Gartz has a patent on the process, and claims he produced 4-HO-DET this way, so I would try to replicate the patented process exactly as he did...
Regardless, please let us know if you actually attempt this, as well as the result.
-E. Borodin
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Everything
(~} ;-}
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Okay, i will try with DET sometime in the future. However i don't have a source for it right now so i dunno, i'll get it figured out.
What about tryptamine? I also read someone successfully grew some more potent shrooms with were supposedly 2-3x as strong as normal.
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Coincidentiaoppositorum
deep psychedelic
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22235008 - 09/13/15 11:36 PM (8 years, 7 months ago) |
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The biosynthetic pathway for psilocybin starts from tryptophan which must be decarboxylated to tryptamine and so on, so a high tryptophan substrate would help, maybe even tryptamine would be bennificial, but I honestly cant say.
-E. Borodin
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Toadstool5
A Registered Mycophile
Registered: 01/22/15
Posts: 1,359
Loc: The Golden State
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22235175 - 09/14/15 12:48 AM (8 years, 7 months ago) |
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Quote:
Biosynthesis of Psilocybin Part II.*Incorporation of labelled tryptamine derivatives.
Stig Agurell and J. Lars G. Nilsson
Departments of pharmacognosy and chemistry, royal pharmaceutical institute, kungstensgatan 49, stockholm VA, Sweden.
Quote:
tryptamine which is readily formed from tryptophan by P. Cubensis, serves as a better precursor than tryptophan. If tryptamine is methylated to N-Methyltryptamine, this is an even better progenitor of psilocybin with incorporations showing that more than half of the psilocybin was derived from the introduced labelled precusor
.
More information of the ability of P. Cubensis to convert tryptophan to tyrptamine is also available from Agurell, S. and Nilsson, L. in the Acta Chemica Scandinavica 22 (1968)
Tryptamine works alright, tryptophan almost not at all, and NMT is the best hands down.
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If you do not know where the mushroom products you are consuming are grown, think twice before eating them. 
- Paul Stamets
AMU Teks  Stro's Write Ups
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22235226 - 09/14/15 01:41 AM (8 years, 7 months ago) |
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Quote:
Toadstool5 said:
Quote:
Biosynthesis of Psilocybin Part II.*Incorporation of labelled tryptamine derivatives.
Stig Agurell and J. Lars G. Nilsson
Departments of pharmacognosy and chemistry, royal pharmaceutical institute, kungstensgatan 49, stockholm VA, Sweden.
Quote:
tryptamine which is readily formed from tryptophan by P. Cubensis, serves as a better precursor than tryptophan. If tryptamine is methylated to N-Methyltryptamine, this is an even better progenitor of psilocybin with incorporations showing that more than half of the psilocybin was derived from the introduced labelled precusor
.
More information of the ability of P. Cubensis to convert tryptophan to tyrptamine is also available from Agurell, S. and Nilsson, L. in the Acta Chemica Scandinavica 22 (1968)
Tryptamine works alright, tryptophan almost not at all, and NMT is the best hands down.
Hmm....I can understand how saving the fungi steps would be bennificial, so I can see how tryptamine would be better, but the fungi needs tryptophan to decarboxylate to produce the tryptamine in the first place, so tryptophan should be just as good, it would just take longer...but if this is not the case please elaborate, because I fully acknowledge I could be missing something here...
-E. Borodin
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Coincidentiaoppositorum
deep psychedelic
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Is it because the NMT is directly methylated and hydroxylated where the tryptophan may be in intermediary States at the time of harvest?
-E. Borodin
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Everything
(~} ;-}
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DAMN OKAY! so this is great this great!
SOMEONE could just get their NMT from someone who extracts DMT from Acacia Confusa Rootbark....we could somehow separate the NMT from the DMT and extract both simultaneously!
Then we could use the NMT in the mushroom grow, so when is the best time to add the NMT? the first poster said i should not put the substituted tryptamines in the substrate??? should i just ignore that completely if i am using NMT?
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bodhisatta
Smurf real estate agent
Registered: 04/30/13
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22235481 - 09/14/15 06:12 AM (8 years, 7 months ago) |
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It would be 100% times easier to just grow good mushrooms
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Coincidentiaoppositorum
deep psychedelic
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Re: Tryptamines in substrate with DPT? [Re: bodhisatta]
#22235641 - 09/14/15 08:22 AM (8 years, 7 months ago) |
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...or to just take good DPT, or use the acacia for huasca brews...
-E. Borodin
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Toadstool5
A Registered Mycophile
Registered: 01/22/15
Posts: 1,359
Loc: The Golden State
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Quote:
saving the fungi steps would be beneficial, so I can see how tryptamine would be better, but the fungi needs tryptophan to decarboxylate to produce the tryptamine in the first place, so tryptophan should be just as good, it would just take longer...but if this is not the case please elaborate, because I fully acknowledge I could be missing something here...
That is a very good observation. It was unexpected and the authors could not figure out why but assumed it had to do with solubilities and enzyme regulation mechanisms.
If you add tryptophan it doesn't help as much as tryptamine because tryptamine concentration downregulates the production of more tryptophan. If you add tryptamine it skips the decarb and goes straight to methyltransferase but DMT is a strong downregulator of tryptamine production so tryptamine is only slightly better than tryptophan because of the skipping of the decarb. They are both downregulated at about the same rate.
If you add NMT it is more soluble and skips the two major downregulations of the pathway, DMT doesn't seem to be as absorbed as well as NMT though so it doesn't help much even though you would think it is the best.
Quote:
Tryptophan is decarboxylated via an enzyme called tryptophan decarboxylase to form tryptamine. This is the last major step in the pathway that is significantly inhibited by a self-feedback downregulation mechanism, meaning that if too much of a particular substance is in the cells the enzyme will stop converting tryptophan to tryptamine. Here is a list of some inhibitors of tryptophan decarboxylase (copied from a paper at the lycaeum; unknown URL:)
http://www.shroomery.org/6227/The-Metabolic-Pathway-of-Psilocybin-Production
Quote:
we could somehow separate the NMT from the DMT and extract both simultaneously!
Then we could use the NMT in the mushroom grow, so when is the best time to add the NMT? the first poster said i should not put the substituted tryptamines in the substrate??? should i just ignore that completely if i am using NMT?
It might be difficult to separate NMT from DMT, maybe column chromatography? Physical separation would be probably be needed.
It has been awhile since I read the papers but i believe they added the chemicals to the bulk substrate, you do not want to autoclave or PC them so either at inoculation or at spawning is ideal.
Spawning is closer to the idiophase when secondary metabolites are booming and when we give them more oxygen to respirate and fruit so i would personally add them to pasteurized CVG and spawn my grains to the supplemented stuff in trays. Let it colonize, consolidate for a couple days, case, allow to colonize another day or two, then fruit.
Edited by Toadstool5 (09/14/15 03:35 PM)
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Everything
(~} ;-}
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22237273 - 09/14/15 03:47 PM (8 years, 7 months ago) |
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thanks everyone! this is some juicy information! okay now i need to figure out how to get my NMT and trptamine.
I am also still really fascinated with creating other substituted amphetamines (lol i mean tryptamines,just woke up) so i am going to read Gartz patent or whatever and see how he accomplished this. "mush" appreciation to all of you!
Edited by Everything (09/14/15 03:48 PM)
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Everything
(~} ;-}
Registered: 06/26/10
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Re: Tryptamines in substrate with DPT? [Re: bodhisatta]
#22237282 - 09/14/15 03:49 PM (8 years, 7 months ago) |
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Quote:
bodhisatta said:
It would be 100% times easier to just grow good mushrooms
I am aware of this, and want to do this too my friend 
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Toadstool5
A Registered Mycophile
Registered: 01/22/15
Posts: 1,359
Loc: The Golden State
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22237371 - 09/14/15 04:06 PM (8 years, 6 months ago) |
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If you do add a substituted tryptamine to the substrate take a fruit body, dry it, grind it up, capsulate it, and send it to ecstasydata.org to be tested. Tell them it is a suspected substituted tryptamine not mdma and in a few weeks to months you will have analytical proof of it working 
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If you do not know where the mushroom products you are consuming are grown, think twice before eating them.
- Paul Stamets
AMU Teks Stro's Write Ups
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Everything
(~} ;-}
Registered: 06/26/10
Posts: 5,157
Last seen: 6 years, 7 months
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22237467 - 09/14/15 04:32 PM (8 years, 6 months ago) |
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I was wondering how i was going to conclude my experiment!
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micro
bunbun has a gungun
Registered: 05/09/03
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22237930 - 09/14/15 06:17 PM (8 years, 6 months ago) |
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Quote:
Toadstool5 said:
That is a very good observation. It was unexpected and the authors could not figure out why but assumed it had to do with solubilities and enzyme regulation mechanisms.
If you add tryptophan it doesn't help as much as tryptamine because tryptamine concentration downregulates the production of more tryptophan. If you add tryptamine it skips the decarb and goes straight to methyltransferase but DMT is a strong downregulator of tryptamine production so tryptamine is only slightly better than tryptophan because of the skipping of the decarb. They are both downregulated at about the same rate.
You are right in that it is a self-feedback mechanism. Tryptophan decarboxylase is highly influenced by the amount of tryptophan so adding it (or any precursor) is counterproductive (or a waste of time). I'm not sure how much solubility matters as there are plenty of insolubles the mycelium can digest, so if it's in the substrate (not necessarily in aq. form) I would think it is safe to assume "it's there."
Looking back at that paper I wrote (many years ago) I posted a chart I found; it shows tryptamine having 62% inhibition and 5-HO-Tryptamine as having 45%. 5-Methoxy-N,N-dimethyltryptamine, 5-Methoxytryptamine and Indole-3-pyruvic acid have 0% inhibition. Those would obviously be interesting to try since you could theoretically get more potency from them. N,N-dimethyltryptamine had 65% which is very close to 62%; given realistic accuracy, one could infer they are the same and the inhibition is simply from the demethylated counterpart in the surrounding cytoplasm. If DPT is handled in the same manner you'll likely see the same thing, otherwise you will get nothing or maybe 4-HO-DPT or even DPT dihydrogen phosphate; don't know. Also, if memory serves me correctly, the psosphorylation part was severely inhibited by tryptamine so there was a disproportionate amount of psilocin vs. -cybin present in the fruitbodies.
You should be able to test for the presence specific compounds pretty easily through various extraction methods and indicators. If you are looking for precise amounts, extraction and then GC/MS is the way to go (and necessary for novel stuff as I doubt that place has the resources to figure out what it is, unless they can provide a printout). Also, even with that it should be extracted for alkaloids first at least, I'd think, unless they do that... I really have no idea about that site. Never used it.
Sorry if that was vague but I obviously don't know what will happen since I've never done it Just a brain dump. As for the 4-hydroxylation, it is interesting but I think it is a bit presumptuous of the authors to claim *anything* will be. I don't think they could realistically come to that conclusion unless they *tested* everything. It is interesting though; it obviously thinks it belongs on that pathway. Well, metaphorically speaking, as it is just an enzyme.
EDIT: Also, I have a large amount of 3-methyl-indole or about 25g straight indole I could give out if anyone wants to use them.
--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)
Edited by micro (09/14/15 06:26 PM)
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Coincidentiaoppositorum
deep psychedelic
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22238302 - 09/14/15 07:41 PM (8 years, 6 months ago) |
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http://www.ncbi.nlm.nih.gov/pubmed/17262409
http://countyourculture.com/2012/02/17/biosynthesis-4-substituted-tryptamine-derivatives/
(For sure go to this link, great stuff about gartz and the results of his biotransformstion experiments...)
Mycelial cultures of Psilocybe cubensis capable of forming psilocybin and psilocin de novo display a high capacity for hydroxylation of tryptamine derivatives at the 4-position. A specific biotransformation of added synthetic N,N-diethyl-tryptamine was found. Thus high amounts of 4-hydroxy-N,N-diethyltryptamine (up to 3.3%) and a minor quantity of 4-phosphoryloxy-N,N-diethyltryptamine (0.01-0.8%) were isolated from fruiting bodies of Psilocybe cubensisin corresponding experiments. This is the firstexampleof a directed biosynthesisof tryptamine substances by fungi. An effective biotransformation of N-methyltryptamine was also demonstrated with surface cultures of Psilocybe semilanceata. Baeocystin, a possible natural precursor of psilocybin, was detected and quantified in the biomasses. No alkaloids could be found in the culture medium.- http://www.ncbi.nlm.nih.gov/pubmed/2614674
Quote:
Sorry if that was vague but I obviously don't know what will happen since I've never done it Just a brain dump. As for the 4-hydroxylation, it is interesting but I think it is a bit presumptuous of the authors to claim *anything* will be. I don't think they could realistically come to that conclusion and 4-phosphorlate
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Coincidentiaoppositorum
deep psychedelic
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Sorry that post got all jumbled, technical issues with my device...
-E. Borodin
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Coincidentiaoppositorum
deep psychedelic
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Abstract Mycelial cultures of PSILOCYBE CUBENSIS, with the ability to form psilocybin and psilocin DE-NOVO, also hydroxylated and methylated fed tryptamine to give psilocin in up to 3.3% dry mass of the obtained fruit bodies. By using HPLC and TLC, it was found that these mushrooms contain only a small amount of psilocybin (0.01-0.2% dry mass). The values of psilocin are the highest described in any mushrooms. - http://www.ncbi.nlm.nih.gov/pubmed/17262409
This says the tryptamine inoculate substrate produced cataphores with low psilocybin, but the highest psilocin rate observed
(Psilocybin is a pro-drug, the 4-phosphorloxy group is hydroxylated and it becomes psilocin in vivo shortly after ingestion....so low psilocybin is not bad, however psilocybin is more stable as far as degradation...regardless very interesting stuff...
I always figured a substrate high in tryptophan would be sufficient....thanks for getting into this topic, I have actually learned a good deal.
-E. Borodin
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Coincidentiaoppositorum
deep psychedelic
Registered: 10/27/14
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Last seen: 8 years, 6 months
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It drives me crazy to talk about biosynthetic pathways when I cant see whats actually going on, so for all you other visual learners out there, I hope this helps.
-E. Borodin
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Toadstool5
A Registered Mycophile
Registered: 01/22/15
Posts: 1,359
Loc: The Golden State
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22238504 - 09/14/15 08:30 PM (8 years, 6 months ago) |
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Quote:
solubility matters as there are plenty of insolubles the mycelium can digest
Solubility is a bad word for it. I'm referring to how well the precursors are absorbed by the mycelium. Perhaps permeability is a better word?
If only there was more literature on this subject 
Great diagram on the pathway!
--------------------
If you do not know where the mushroom products you are consuming are grown, think twice before eating them.
- Paul Stamets
AMU Teks Stro's Write Ups
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Coincidentiaoppositorum
deep psychedelic
Registered: 10/27/14
Posts: 1,965
Last seen: 8 years, 6 months
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22238673 - 09/14/15 09:10 PM (8 years, 6 months ago) |
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Here's some links that may be helpful, ill be more willing to discuss my veiws on the matter in the morning when I have got some rest.
http://www.lycaeum.org/mv/mu/cubensis_parameters.html
this link is great general information, really good stuff, highly recommended.
http://www.researchgate.net/publication/6542579_Biotransformation_of_Tryptamine_in_Fruiting_Mycelia_of_Psilocybe_cubensis
Biotransformation link.
http://onlinelibrary.wiley.com/doi/10.1002/jobm.3620290608/abstract;jsessionid=C4069DF287E79A79E4B17E9DDB0708E9.f02t02
Biotransformation of tryptamine derivatives in mycelial cultures (psilocybe cubensis) by gartz
onlinelibrary.wiley.com/doi/10.1002/jobm.3620290608/pdf
http://chemistry.mdma.ch/hiveboard/tryptamine/000127125.html
Stuff from the hive on the topic
http://www.stainblue.com/ah.html
Here's some hoffman information
https://www.erowid.org/archive/rhodium/pdf/ psilocybin.biosynthesis-2.pdf
And an erowid PDF on psilocybin biosytnthesis.
https://www.erowid.org/culture/characters/gartz_jochen/gartz_jochen.shtml
here's erowids gartz page, there's some great links in it
https://www.thevespiary.org/talk/index.php?topic=12665.10;wap2
(Everything below came from the link.above)
Psilocybin biosynthesis 4-Hydroxylated or 4-methoxylated indoles are very rare in nature. The only known examples beside the psilocybin-type alkaloids are the 4-hydroxylation of indole acetic acid by Aspergillus niger strains (54), methoxylated b-carbolines from Banisteriopsis argentea and Picrasma javanica, the reserpine analog venenatine from Alstonia venenata, the yohimbine analog mitragynine from Mitragyna speciosa (79, 82 p. 703), and the aminopyrimidyl-indolic meridianines from the tunicate Aplidium meridianum (25). Psilocybin has a simple structure in contrast to the former alkaloids. It is formally derived from tryptophan in five distinct biosynthetic reactions, i.e. decarboxylation, indole-hydroxylation, two N-methylations, and O-phosphorylation (Table 1). Feeding experiments with putative intermediates, analogs of them, or radioactive precursors supported the view that tryptophan decarboxylation is the first biosynthetic step and that O-phosphorylation is the final step. The sequence of the remaining intermediate reaction steps is still unclear. Some authors even suppose a biosynthetic grid with multiple routes to psilocybin (Figure 1) (13, 5, 78).
Abbreviation Full name Decarboxylase Tryptophan Decarboxylase Hydroxylase Tryptamine 4-Monooxygenase, Tryptamine : Oxygen Oxidoreductase, 4-hydroxylating Methylase Tryptamine-w-amino-methyltransferase Phosphorylase Psilocin-O-phosphotransferase
Table 1. The psilocybin synthesizing enzymes. The full name follows standard enzyme nomenclature while the abbreviated name is used throughout this text. The enzymes may actually consist of several related enzyme with slightly different substrate specifities.
Expression cloning procedure For a definitive evaluation of psilocybin biosynthesis, the participating enzymes need to be isolated and tested for their substrate specifities and activities. The feasible way to do this was by genetically cloning and heterologous expression. The cloning methodology chosen is an alternative to PCR based procedures and has been devoloped and used succesfully to clone a broad range of fungal enzymes (21, 20, 15, 14, 53). It consists of ligating a cDNA library into an expression vector (pYes2), transforming a host organism (S. cerevisiae), and a screening the resulting colonies for enzyme activity (Figure 2). The success of such expression cloning procedures depends on reliable and sensitive enzyme assays for the colony screening step. Indeed it was possible to find such assays for all enzymes of the psilocybin biosynthesis pathway (Table 2). A second important requirement is the use of biomaterial containing high amounts of the respective enzymes mRNA. Usually this is a growing tissue containing the metabolites of interest. In this study still developing Psilocybe tampanensis sclerotia were used for mRNA extraction. They grew rapidly and reliably on a special medium and contained high amounts of psilocybin and psilocin.
Total RNA isolation from enzyme producing biomaterial \/mRNA preparation \/cDNA synthesis \/Ligation into E. coli / S. cerevisiae shuttle expression vector (pYes2) \/Amplification in E. coli \/Colony pooling and plasmid preparation (20 pools of 5,000 colonies each) \/Yeast transformations \/\/\/\/Enzyme activity screenings of colonies by color reactions after inducing expression (galactose) \/Rescreening positive clones \/Cross-transformation of E. coli, insert sequencing and analysis
Figure 2. Expression cloning flow scheme. As used by Dalbøge et al. to clone a broad range of fungal enzymes.
Enzyme Screening procedure Decarboxylase 5-fluoro-tryptophan resistance Hydroxylase feeding tryptamines, Keller's reaction Methylase feeding tryptamines, derivatization and removal of substrate, radioactive detection Phosphorylase feeding psilocin, derivatization of substrate, Keller's reaction after phosphatase treatment
Table 2. Enzyme activity screening procedures. All tests can be performed on colonies to allow a parallel screening procedures. The Keller's reaction is a very sensitive and specific color reaction for hydroxylated indoles.
Mushroom media and culturing Media ingredients and sources: dried unrefined sugar-cane extract (Rapadura, organic food or third world stores), sugar-beet syrup (75% dry matter, food stores), mixed pollen (organic or health food stores), dry yeast extract (Oxoid, England), peptone (Difco Laboratories), agar (Merck, Germany), commercial malt extract / yeast solutions "Salvator" (containing 18.3% stammwuerze) and "Hefe Weissbier dunkel" (dark unfiltered wheat beer, containing 12.4% stammwuerze, both from Paulaner, Munich, Germany). The mycelia were cultured on Parafilm sealed MEY plates (6% malt extract syrup, 0.6% yeast extract, 1.5% agar) at 28°C ± 2°C in an incubator (6). For propagation 1 cm x1 cm blocks were cut out and transferred to the middle of a new agar plate under sterile conditions. Plant hormones were added as ethanolic solutions. Media containing KH2PO4, pollen, acetic acid, citric acid, and ascorbic acid medium were autoclaved after adjusting the pH. Media from beer (pH around 5.5) were autoclaved for 40 min.
Mushroom extraction For a simple but efficient extraction of psilocin as well as psilocybin the following method was applied (98, 58, 45). 7.5 mg lyophilized mushroom material was homogenized in a 1 ml glass mortar with 250 µl methanolic extraction solvent (75% MeOH, 0.1% ascorbic acid). This suspension and 2 x125 µl methanolic rinsings were pooled in a microcentrifuge tube. After agitation for 10 min at RT the tube was centrifuged for 2 min at 14,000 rpm at RT (5415C centrifuge), the supernatant was transferred to a fresh tube, and the pellet was resuspended in 250 µl ethanolic extraction solvent (75% EtOH, 0.1% ascorbic acid). After agitation for 10 min at RT and centrifugation as above the supernatants were pooled and stored at -20°C in an airtight microcentrifuge tube.
Reagents and procedures from the cDNA Synthesis System Kit (Gibco BRL) were used in combination with a vector primed cDNA synthesis literature method (85). This approach was not succesful.
Here's a cool transformant screening procedure; 5-Fluoro-tryptophan decarboxylase screening For tryptophan decarboxylase screening transformed FY 73 cells were plated directly onto SC-gal containing 0.5 - 1.5 mM 5-fluoro-tryptophan and were incubated up to 6 days at 30°C.
No-Go for submerged culture; The tested Psilocybe strains showed variable preferences for the tested media. In general, the defined Leatham’s media, sugar-cane medium, and beer-based media were poor substrates compared to malt extract based media. The submerged culture produced the highest amounts of biomass, followed by the surface cultures on soft agar (0.2%). But under both conditions the mycelia did not produce measurable amounts of alkaloids. The same was true for Ps. tampanensis submerged cultures in 6% malt extract with no supplement, 0.3% yeast extract, 2% pollen, or both added (data not shown).
Ps. cubensis, Ps. tampanensis, Ps. azurescens, and Ps. cyanescens were grown in different media. After 14 days the mycelium was harvested, weighed, extracted, standardized, and analyzed for psilocin and psilocybin content. Ps. cubensis produced psilocin, but no psilocybin under the conditions analyzed. In contrast Ps. tampanensis and Ps. azurescens produced both alkaloids. Ps. azurescens and Ps. cyanescens were growing very slowly on all media tested.
Psilocin and psilocybin Rf values Psilocin and psilocybin references (in slightly acidic solution, isolated from Ps. cyanescens fruiting bodies) were TL-chromatographed using various solvent systems. The plates were stained with Van Urk’s reagent (DMCA modification) and Rf values were observed (Table 8).
Solvent system Psilocin Rf Psilocybin Rf H2O: MeOH: AcOH (50 + 50 + 1) 0.92 0.88 MeOH 0.54 0.11 Acetone 0.30 0.06 MeOH: AcOH (1 + 1) 0.33 0.13 H2O 0.88 0.47 H2O: AcOH (1 + 1) 0.88 0.76 nPrOH: H2O: AcOH (10 + 3 + 3) 0.71 0.39 CH2Cl2 0.00 0.00
-E. Borodin
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Coincidentiaoppositorum
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I'm not sure how specific any of that information was, but it kept me interested.
-E. Borodin
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micro
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Quote:
Coincidentiaoppositorum said:
Mycelial cultures of Psilocybe cubensis capable of forming psilocybin and psilocin de novo display a high capacity for hydroxylation of tryptamine derivatives at the 4-position.
High capacity =/= everything. I guess it was just taken out of context.
They have isolated those enzymes? I wonder if they have been sequenced or just ID'ed on western blot or something and then named?
Only reason I ask is it would be funny to TA-clone them into S. cerevisiae, assuming one had standard molbio resources and the ability to purchase the primers and a kit (if they aren't stocked). It would take me half a day (mostly waiting) to run the PCR and prolly get the rest done that day, culture overnight, transfer to TB/LB media, etc. I don't work in molbio anymore, though.
EDIT: Oh wait, looks like they did that.
I want some culture of that 
Interesting info; I'll have to check it out in depth when I have a bit.
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22240628 - 09/15/15 10:19 AM (8 years, 6 months ago) |
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The biosynthesis of psilocybin in psychedelic mushrooms is a multi-step process, and the precise
mechanism is debated by many authors...-countyourculture
Experiments with radiolabled precursors have shown that this is likely the primary path to psilocybin,however, labelled 4-hydroxytryptamine was also shown to be incorporated into the produced psilocybinindicating the possibility of an additional biosynthetic pathway. Other alkaloids present in psilocybin mushrooms such as baeocystin or norbaeocystin are not explained by this single pathway as well.
An elegant alternative has been proposed. What if instead of a single path and a set order of modifying reactions, there were multiple paths to psilocybin – with branching edges that led to baeocystin and norbaeocystin? The enzymes would compete and feed back among each other in a biosynthetic grid that preferred to produce psilocybin and psilocin but also produced small amounts of baeocystin andnorbaeocystin as typically seen in nature. -countyourculture
They are refering to this pathway:
-tryptophan--tryptamine--DMT-psilocin-psilocybin
I know dimethyltryptamine better than psilocin...so ill start with what I know about its enzamatic pathway...
Which starts from tryptophan which is decarboxylated (amino acid decarboxylase, AADC)
Next we have tryptamine which is met with indole-amine methyl transferase (INMT) and exposed to s-adenosyl-methionine. (SAM) which becomes s-adenosyl-l-homocystine (SAH) as it adds the methyl grouping.
First time we get NMT, second round DMT...
Then its enzamatic hydroxylation then 0-phosphorlation to psilocybin
As far as isolation of the enzymes, yes, it has happened, and work is being done. But molecular biology is not my strong point, I'm around 3rd year organic chemistry in my knowledge and skill level here, which means anything in TIHKAL or PIHKAL is a breeze...it gets a bit more complicated with Albert hoffman as far as techniques and equipment goes, but its still nothing a second or third year organic chemistry student couldn't figure out.
And that's where I spend most my time, in the study of Sasha shulgin, Albert hoffman, David E. Nichols, Daniel Trachsel and even some of the ethnobotanists like schultes or wasson, and I love to understand how these things are acting in our body or formed in nature, but in all honesty its the organic chemistry work of people like shulgin, heffter, Nichols, hoffman, etc..that fascinates me, and that relates to my educational goals.
It looks like you have your work cut out for you though, molecular biology is a very demanding field of study, I have not looked into it much, well any more than I would have to, and since I'm a person who enjoys recipe oriented activities and math, org. Chem just seemed to really fit, where biology, physiology, and things of this nature, seemed like things I had to learn because of their relation to something I cared about, and I see them more as a pain in the ass than fun, but that's why science is divided into specialized fields.
So sorry, I wish I could help, but your getting a bit out of my range of study, and though it does involve tryptamine chemistry, there's probably around 100 different active tryptamines just covered by TIHKAL alone, so ive only gotten really in depth on a few of them, and I do know a good deal about psilocybin, but its just one of many, plus I have only been in study of these compounds for 2.5-3 years, I still have a good deal to learn.
Orbitals are for mathematicians, organic chemistry is for people who like to cook. -Alexander shulgin
-E. Borodin
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micro
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AFAIK, it's debated as much past tryptophan decarboxylase. I think that was from Gewirtz et al. but I don't remember; it would be easy enough to test -- all you'd need is to add glyphosate (Round Up) to the substrate and it should stop producing alkaloids.
I'm not in molbio anymore. I moved to San Francisco and sold out. I've been a software developer for the last decade or so... The pay is a lot better though :3
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22241074 - 09/15/15 12:03 PM (8 years, 6 months ago) |
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Quote:
micro said:
AFAIK, it's debated as much past tryptophan decarboxylase. I think that was from Gewirtz et al. but I don't remember; it would be easy enough to test -- all you'd need is to add glyphosate (Round Up) to the substrate and it should stop producing alkaloids.
I'm not in molbio anymore. I moved to San Francisco and sold out. I've been a software developer for the last decade or so... The pay is a lot better though :3
Regardless your a smart person, the psychedelic community needs all the intellegent individuals it can get, even if your contributions are theorys in a notebook or things you publish so who ever can do the most with them will find the information, it would be a waste to not try to share the things you know, specially in a field like psychedelics, where there is no college classes and where misinformation is rampant.
-E. Borodin
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Toadstool5
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Quote:
glyphosate (Round Up) to the substrate and it should stop producing alkaloids.
On the subject of inhibiting enzymes. Does anyone know what catalyzes the indole-4-monooxygenase for the hydroxylation? I assume it is some cytochrome P450 and was wondering if it can be inhibited without causing other issues with the metabolism?
Any help would be super duper appreciated 
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micro
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22244957 - 09/16/15 09:14 AM (8 years, 6 months ago) |
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Quote:
Toadstool5 said:
Quote:
glyphosate (Round Up) to the substrate and it should stop producing alkaloids.
On the subject of inhibiting enzymes. Does anyone know what catalyzes the indole-4-monooxygenase for the hydroxylation? I assume it is some cytochrome P450 and was wondering if it can be inhibited without causing other issues with the metabolism?
Any help would be super duper appreciated
K, now I'm lost.
a.) What context the quote has, and
b.) are we talking about mushroom metabolism or the metabolism of mushrooms (by people) now?
Reason I asked is you mentioned cytochrome P450. I can't imagine you were asking about metabolic pathways in mycelium or you obviously answered your own question. If you're not, well... I really don't know what we're talking about in the first place.
The two kind of contradict :V
Just to clarify: When I hear "cytochrome P450" I generally think "drug interactions."
But, since (I hope) we are talking about mycelial production of alkaloids you answered your own question -- it's an enzyme, which *is* the catalyst.
Maybe I'm missing something.
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Edited by micro (09/16/15 09:46 AM)
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Toadstool5
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22249015 - 09/17/15 12:20 AM (8 years, 6 months ago) |
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In the context of Mushroom metabolism.
As far as I understand things:
There are many different cytochrome p450 enzymes. I can only find information on tryptophan 5-monooxygenase and tryptophan 5-hydroxylation and even less on their inhibition.
I do not know if a tryptamine 4-monooxygenase Cytochrome p450 enzyme exists and if inhibiting it will cause other imbalances with different cytochrome p450 type enzymes within the mycelium.
I assume the 4-hydroxylation of indoles normally involves the same type of cytochrome P450 dependent monooxygenases as the 5-hydroxylation. The problem is, I don't know what can be done to inhibit the 4-monooxygenase and 4-hydroxylase without inhibiting other enzymes and whether or not techniques for the inhibition of 5-monooxygenase and 5-hydroxylase work.
Glyphosate for example, would work very well to inhibit whatever cytochrome p450 type monooxygenase and hydroxylase is causing the reaction but it would also shut down many other cytochrome p450 enzymes
So
1.Is there a specific Cytochrome P450 protein involved in the tryptamine 4-monooxygenase/hydroxylation that anyone knows of?
2. Can it be inhibited using the same methods for the tryptamine 5-monooxygenase/hydroxylation cytochrome p450 proteins?
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22249763 - 09/17/15 07:07 AM (8 years, 6 months ago) |
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Their inhibition? Mono amine oxidase inhibitors should inhibit the molecules metabolism for a moment as the molecules are monoamines...I once took 3g peganum harmala with 3g stropharia cubensis, and it was overwhelmingly intense and long lasting...
Otherwise
www.maps.org/research-archive/w3pb/2002/2002_Passie_22704_1.pdf
http://www.ncbi.nlm.nih.gov/pubmed/14578010
Maybe these will help, I still don't understand fully what your asking about inhibition of metabolism though...
-E. Borodin
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micro
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Glyphosate inhibits the Shikimic Acid Pathway.
I don't know the specifics about the enzymes involved. I'm not sure if it is even known.
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Toadstool5
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22251799 - 09/17/15 03:36 PM (8 years, 6 months ago) |
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MAOI's inhibit the oxidation of the N-H bonds in amines so I don't believe they would help prevent the C-H oxidation at the 4 position.
Quote:
Glyphosate inhibits the Shikimic Acid Pathway.
I don't know the specifics about the enzymes involved. I'm not sure if it is even known.
Glyphosate would work great but i want the shikimate pathway to work up to the point of tryptamine/NMT/DMT synthesis and then stop it at the tryptamine monooxygenase/hydroxylation so the mushrooms only produce tryptophan/tryptamine/NMT/DMT.
It seems that the exact enzymes for the tryptamine-4-monooxygenase and tryptamine-4-hydroxylase aren't known yet so I will probably use some of the methods used to inhibit the tryptamine-5-monooxygenase and tryptamine-5-hydroxylase and see if psilocybin, psilocin, or baeocystin are still produced.
Do you guys see where i'm getting at here? If you can inhibit the enzyme(s) responsible for the oxidation of tryptamine, NMT, and DMT then in theory DMT can be produced from filamentous fungi without genetic modification. Most sources are biological or GMO.
We need more info on the 4 position mechanism, how it relates to and differs from serotonin and the 5 position mechanisms in particular.
Look at all the speculation over 4-hydroxy-5-methoxydimethyltryptamine and lack of experimental data 
This is one article I am really interested in applying to psilocybian species to prevent the 4-hydroxylization of tryptamine, NMT, and DMT:
Quote:
A Putative Metabolite of Serotonin, Tryptamine-4,5-Dione, Is an Irreversible Inhibitor of Tryptophan Hydroxylase:  Possible Relevance to the Serotonergic Neurotoxicity of Methamphetamine
Monika Z. Wrona and Glenn Dryhurst
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Chemical Research in Toxicology
Vol. 14: Issue. 9: Pages. 1184-1192
Publication Date (Web): August 4, 2001
DOI: 10.1021/tx010037c
Tryptamine-4,5-dione (T-4,5-D) is formed as a result of oxidation of 5-hydroxytryptamine by superoxide (O2-·), nitric oxide (NO·), and peroxynitrite (ONOO-). T-4,5-D rapidly inactivates tryptophan hydroxylase (TPH), derived from rat brain, probably as a result of covalent modification of active site cysteine residues. The activity of TPH exposed to T-4,5-D cannot be restored by anaerobic reduction with dithiothreitol (DTT) and ferrous iron (Fe2+) indicating that the inactivation is irreversible. 7-S-Glutathionyl-tryptamine-4,5-dione, formed by the rapid reaction between T-4,5-D and glutathione, also inhibits TPH but in this case the activity is restored by anaerobic reduction with DTT/Fe2+. The results of this investigation may be relevant to the initial reversible and subsequent irreversible inactivation of TPH evoked by methamphetamine and 3,4-methylenedioxymethamphetamine.
Edited by Toadstool5 (09/17/15 03:53 PM)
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micro
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22252141 - 09/17/15 04:57 PM (8 years, 6 months ago) |
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RNAi
edit: that's assuming you have it sequenced
otherwise, maybe look for self-feedback downregulation mechanisms?
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Edited by micro (09/17/15 05:20 PM)
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22254615 - 09/18/15 06:03 AM (8 years, 6 months ago) |
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So your trying to inhibit hydroxylation or pbospborlysstion to obtain DMT in your final product?
-E. Borodin
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micro
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4-OH
At least, that's what I got out of it.
I wasn't even aware anything was sequenced yet, until your post.
Then again, I've been out of the loop for like a decade or so 
edit: dyslexia *rolleyes*
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Edited by micro (09/18/15 10:12 AM)
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22255612 - 09/18/15 10:37 AM (8 years, 6 months ago) |
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4-HO-DMT is psilocin...
This is why this is all confusing to me, it seems like several different things have been jumbled together in a way to make it as unclear as possible, I'm interested in the topic here, but I'm having trouble trying to decipher your intent.....meaning I generally know the processes and enzymes here, but its hard for me to tell what exactly your trying to say.
There's a grid for the biosynthetic pathway of psilocybin because after tryptophan decarboxylation its unclear what pathways the fungi are using, there are a few diffferent possibilities...
It sounded to me like you were asking if by inhibiting hydroxylation and Phosphorylation of the dimethyltryptamine if you could yeild it in the fruiting bodies...
But I got somewhat lost...
-E. Borodin
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micro
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Quote:
Coincidentiaoppositorum said:
4-HO-DMT is psilocin...
This is why this is all confusing to me, it seems like several different things have been jumbled together in a way to make it as unclear as possible, I'm interested in the topic here, but I'm having trouble trying to decipher your intent.....meaning I generally know the processes and enzymes here, but its hard for me to tell what exactly your trying to say.
There's a grid for the biosynthetic pathway of psilocybin because after tryptophan decarboxylation its unclear what pathways the fungi are using, there are a few diffferent possibilities...
It sounded to me like you were asking if by inhibiting hydroxylation and Phosphorylation of the dimethyltryptamine if you could yeild it in the fruiting bodies...
But I got somewhat lost...
-E. Borodin
So, if the 4-hydroxylation were inhibited it might make DMT...
Or, it might skip over it entirely as it isn't "recognized" as a ligand any more.
Phosphorylation should be easy, just bump up the potency.
It has already been demonstrated this is the case.
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22255750 - 09/18/15 11:10 AM (8 years, 6 months ago) |
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A ligand has to do with binding and binding affinity, it can be binding to other compounds or there can be binding to receptor sites....
.... which is why I'm having trouble following whats going on here, at times I think the biosynthetic pathway for psilocybin is being discussed, at other times its human metabolism of the compound being discussed, and at other times its chemical analysis or growing of the fungi being talked about ...sorry, I got lost at some point.
Sorry, you guys will have to let me figure out what your saying before I can participate productivly...
-E. Borodin
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micro
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The enzyme has at least *some* specificity, right?
I don't know what to better call it.
My point was removing the hydroxyl group could cause the enzymes upstream to lose specificity.
If you can think of a better term I'm all ears.
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Coincidentiaoppositorum
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22256251 - 09/18/15 01:32 PM (8 years, 6 months ago) |
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Your going over my head, I'm trying to keep up though. I'm still learning all this as well.
-E. Borodin
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Toadstool5
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Quote:
So, if the 4-hydroxylation were inhibited it might make DMT...
Or, it might skip over it entirely as it isn't "recognized" as a ligand any more.
Exactly it may create tryptamine, NMT, DMT, or perhaps some novel compounds not yet created.
We are talking about limiting the mushroom's metabolism while growing to yield different tryptamines.
Tryptophan is created in the mushrooms through the shikimate pathway, then it is decarboxylized to tryptamine. At this point it is either methylated by enzymes or oxidized by enzymes to yield the hydroxylized version of the tryptamines.
The proteins associated with the enzymes for the oxidation of the tryptamines and the enzymes themselves are not yet known.
I think they might be similar to other known tryptophan monooxygenase enzymes associated with cytochrome P450 proteins.
If we can inhibit the enzymes chemically without effecting or removing the proteins, we can alter the tryptamines produced by the mushrooms.
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Near Dylan
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22327228 - 10/03/15 08:36 AM (8 years, 6 months ago) |
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Any progress on this experiment yet?
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Toadstool5
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Re: Tryptamines in substrate with DPT? [Re: Near Dylan]
#22328783 - 10/03/15 03:04 PM (8 years, 6 months ago) |
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Nope, i am so poor i don't have the resources to start that type of project. Here and there I will try what i can using the methods to inhibit 5-hydroxy to see if they also have an effect on the 4-hydroxylation.
However, I think it will take more effort by the DIY bio movement to identify the gene responsible before we can hope to manipulate or shut it down.
I'm not educated in the ways of sequencing, transfers, isolation, and all that cool genetic modification stuff either so even if i had the lab and resources I would be going off of secondary research and hail-mary's.
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If you do not know where the mushroom products you are consuming are grown, think twice before eating them.
- Paul Stamets
AMU Teks Stro's Write Ups
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micro
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22329261 - 10/03/15 04:34 PM (8 years, 6 months ago) |
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I read a journal article on this some time after the thread seemed to have died.
DPT *does* in fact work, and gets 4-hydroxylated.
N,N-DMT does not work very well; it causes inhibition of the metabolic pathway at some point.
Funny thing is, there is a similar thread from 2004 and I had posted in it; I just forgot
If you want to inhibit gene expression though, you would need more sophisticated resources.
I think the easiest way these days is with RNAi.
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Toadstool5
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22329732 - 10/03/15 06:43 PM (8 years, 6 months ago) |
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Quote:
I think the easiest way these days is with RNAi.
So true 
I have a feeling a genetics nerd will figure it out eventually and in the meantime it is a waiting game. There isn't enough focus on fungi, you know what I'm talking about micro. Even with something as common as mating types we lack serious research.
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If you do not know where the mushroom products you are consuming are grown, think twice before eating them.
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Re: Tryptamines in substrate with DPT? [Re: Toadstool5]
#22329844 - 10/03/15 07:14 PM (8 years, 6 months ago) |
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Yeah. I'm surprised, too.
You would think with all the money involved in commercial production someone would.
Especially since the mating-type loci control fruitbody production.
I'm hoping once I move I can try some stuff.
I haven't scored a whole lot of popularity with the neighbors playing mad scientist here
The people upstairs actually called the cops and told them I was insane and going to blow everything up.
Of course, the cops just thought she was a fruit loop which is p much the case anyway.
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Toadstool5
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Re: Tryptamines in substrate with DPT? [Re: micro]
#22330731 - 10/03/15 10:56 PM (8 years, 6 months ago) |
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Quote:
The people upstairs actually called the cops and told them I was insane and going to blow everything up.
I know how that goes all too well hahaha
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CraterBait
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Re: Tryptamines in substrate with DPT? [Re: Everything]
#22352917 - 10/09/15 01:00 AM (8 years, 6 months ago) |
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Quote:
Everything said:
Basically, i am going to try this. or if a friend wants to i will give him the DPT to add to substrate.
Not even shulgin has info on dosage of 4-ho-DPT but i have a pretty good guess its around 25mg for a moderate to strong dose
This is a project in the distant future, nothing of now. but will report back if i have results. there is seriously no info on 4-HO-DPT because shulgin says it is so hard to make but i think he meant synthetically not through the mushroom, at the time of pihkal this method was not known.
I know in tikhal, page 247 starts a chapter 'dmt is everywhere' shulgin illustrates the properties of mycellia being able to 4-hydroxylate tryptamines with little bias and issues a few examples. It probably was indeed already known to shulgin.
My question to OP is, to help me understand his experiment: With so little information on 4-HO-DPT how do you plan on confirming its presence in the fungus? Furthermore; how do you intend on isolating it from other substances that may interfere with a bioassay.
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