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Capnine - biofilm bacteria's dirty little secret
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Claudia
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 Posted: Tue Jun 26th, 2007 08:00

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I was looking up "biofilms" and now realise that we have biofilm on our teeth - associated with dental plaque.  Might this start to explain the connection between poor dental health and heart disease & other problems?



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Ruth Goold
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 Posted: Tue Jun 26th, 2007 13:05

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Trevor said:

"I am pretty busy at the moment, but I would be appreciative if you could spare the time to look over supplementary table 5 of Wang, et al, and make sure it correlates with the citation you posted:


The following research group failed to induce cathelicidin in the murine VDR:

Gombart AF, Borregaard N, Koeffler HP. 2005. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.  FASEB J. 19(9):1067-77 PMID 15985530

Again, if you could take a quick look and let me know if there are any significant differences in the outlook of the two groups."

 

Sorry to be slow on this but I did take a look at the results from the three studies and conclude the following:



I think all three studies are consistent with each other and with the idea that, in primates, the VDR is a major transcription factor (TF) involved in the up-regulation of CAMP (cathelicidin antimicrobial peptide), whereas in mice, rats and dogs, the VDR is not a TF for CAMP.  

 However, the general picture emerging is that even in mice and rats, the NHRs, including the VDR,  are major TFs for other classes of AMPs.  In addition, the murine CAMP gene is regulated by one or more of the NHRs (just not directly by the VDR).

 Here is a summary of findings:

Gombart et al.:

Simple and elegant study demonstrating that in a variety of human cells and cell lines, 1,25 D (and some analogues) induce transcription of the CAMP gene and production of the active peptide.  Used DNA sequence analysis to identify a VDR response element (VDRE) in a SINE about 600 base pairs upstream of the VDR transcription start site (TSS).  Cloned the DR3 VDRE into a reporter plasmid and demonstrated its ability to induce expression in cells treated with 1,25 D. 

 Also demonstrated that 1,25 D did not induce transcription of the orthologous C(R)AMP gene nor production of the active peptide in murine bone marrow cells.  Found that there was ‘considerable’ homology between the murine and human DNA sequences upstream of the VDR TSS, but that mice (and rats and dogs) lacked the SINE containing the VDRE.

Did not look for other NHR response elements in the mouse CAMP promoter regions.

Wang et al.:

Performed ‘genome-wide’ DNA sequence scan (humans and mice) for VDREs (both types, DR3 and ER6).  Also did some expression analysis in a human microarray to confirm that putative VDREs were indeed associated with up- or down-regulation of associated genes. 

Microarray analysis revealed 913 genes that were up– or down-regulated by 1,25 D on the human Hu133A microarray (but not CAMP).   However, the authors claim that they showed that 1,25D induced expression of both human CAMP and defensin beta 2 (defb4) genes in SCC25 cells (human squamous carcinoma cell line) in a previous study (Wang et al. 2004).

 Genome sequencing identified a DR3 VDRE upstream of the human CAMP gene (as far as I can tell, this is the same one identified by Gombart et al).  (N.B.  Wang et al. searched further (10 kb upstream of the gene compared to a 1 kb search by Gombart et al.) but apparently identified only the same VDRE).  

Screening of the mouse genome for VDRE sequences, revealed about 3000 orthologous mice and human genes that both had VDREs.  However, the CAMP gene was not one of them (according to Supplementary Table 5 and to the following statement from the study):  

We identified consensus promoter-proximal DR3-type VDREs in the promoters of the [human] defB2 and camp antimicrobial peptide genes, which mediated their induction by 1,25 D.  However, neither the [response] elements nor the regulation by 1,25D appear to be conserved in mouse. 

 OK – now things start to get interesting ….

 Brahmachary et al.

These researchers did no expression work whatsoever – but did do a large-scale genome sequence search for many TF response elements (NHRs as well as others) in the promoter regions of 22 classes of AMPs for which they could identify orthologous genes in human, rats and mice.  They identified 3 classes of TFs (NHRs, liver-specific, and neuron system-specific) that were consistently associated with AMP genes.  NHRs were in the top-ranked TFs for eleven of the 22 AMP families (including cathelicidin).  GR, RXR-alpha, AR, VDR and T3R-alpha were involved in transcriptional control of 20, 18, 17, 16 and 15 of the 22 AMP families (in one or more species).  

In the case of CAMP, the following TFs were identified in the upstream sequences in a composite of three species (mouse, rat, human):

  1. The TF associated with binding of PEA3, c-Ets1, PU.1, LyF-1, c-Ets-2, and NF-AT1, AT2 and AT4
  2. Nkx2-1
  3. Unknown (newly-identified) TF
Overall, NHRs were the most frequent TFs associated with 6 of the 22 AMP families in humans, rats and mice:   alpha defensin, cathelicidin, Dbi, lactoferrin, Spyy and ZAP.

The VDRE was detected in 16 families of AMPs (but not in all species) including the  eight listed in the previous sentence and apoa2, beta defensin, bpi, calgranulin, spli, granulin, hepcidin, mbp, secretogranin and vasostatin.  The VDRE is conserved among humans, rats and mice in some AMP families (e.g. ZAP).

So, I think that everyone is agreement that the VDR does directly induce CAMP expression in humans (and other primates) and does not directly induce CAMP in rats, mice and dogs.  However, other NHRs such as RXR-alpha are important TFs for CAMP in mice.

 
  Ruth

 

Last edited on Tue Jun 26th, 2007 14:20 by Prof Trevor Marshall



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Prof Trevor Marshall
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 Posted: Tue Jun 26th, 2007 14:34

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Excellent summary Ruth. Almost exactly what I concluded for the review paper I just finished:) I did edit the bit about RXR as RXR always heterodimerizes with VDR when working on human CAMP (I think:))

So here is the really, really interesting bits:
If you look at my data to the FDA talk you can see that 1,25-D goes into all the Brahmachary receptors with high affinity - GCR, AR, PR, and alpha-Thyroid. There has to be a very good chance that they all work together in the innate immune system, each with slightly different distribution of tasks. So here are the pretty pictures. First, 1,25-D docked into the GCR pocket alongside Dexamethasone for comparison



and into the Thyroid-alpha alongside T3



Enjoy!
..Trevor..
 

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 Posted: Tue Jun 26th, 2007 15:02

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At the risk of being labeled a dolt, I admit:  I don't understand what these pictures mean.  I've read the whole thread...

Yes, D125 or D25 or VDR is making my immune system malfunction.  But I don't understand the pictures.

With honesty,

Sherry



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Prof Trevor Marshall
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 Posted: Tue Jun 26th, 2007 15:48

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Sherry,
The pictures show that a high level of 1,25-D is capable of displacing cortisol and T3 from the active ligand binding pockets of a portion of those receptors, and inactivating those receptors. Hence those receptors can't make those nice antimicrobial peptides (and other things). Nor can the Thyroid alpha or beta receptors respond to normal quantities of T3 (T3 is made in the body from T4, Thyroxine).

1,25-D would only displace the natural ligands when its concentration rises well above where it would normally be controlled by the VDR. Since the bugs cripple the VDR, the 1,25-D has few limits on its strength, and we see (clinically) the results of it affecting these other receptors.

So although the microbes only evade 16 families of AMP by disabling the VDR, the consequential rise in 1,25-D can disable a bonus 67 families of AMP... Unfortunately, it also makes the host sick...
 

 

Last edited on Tue Jun 26th, 2007 15:55 by Prof Trevor Marshall

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 Posted: Tue Jun 26th, 2007 16:00

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So bacteria disable VDR which contributes to the rise of 1,25-D which disables other receptors ? This is pretty twisted :) And these other receptors are responsible for immune system proper function too ?  I wonder why nature made 1,25-D being able fit other receptors.

Last edited on Tue Jun 26th, 2007 16:02 by wrotek



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Russ
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 Posted: Tue Jun 26th, 2007 16:05

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That's real interesting.  The model is getting clearer and clearer all the time.  Eventually it will be so clear that even the skeptics will be able to see it.

It sounds like the immune system would work better if 1,25D did not have any affinity for those other receptors and only binded to VDR.  Do you think there is an evolutionary reason why 1,25D has affinities for these other receptors?  Or is it just the nature of ligands and receptors that at a high enough concentration a ligand is going to bind to a lot of different receptors?

Thanks!


Edit: Wrotek, just saw your post.  I guess great minds think alike.  Or maybe that should be brain-fogged minds think alike. :)

Last edited on Tue Jun 26th, 2007 16:11 by Russ



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Ruth Goold
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 Posted: Tue Jun 26th, 2007 16:07

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Trevor,
In your answer to Sherry, I think you answered my question ... which was:

When 1,25 D is docked into the other nuclear hormone receptors, is it an agonist or antagonist?

Ruth



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 Posted: Tue Jun 26th, 2007 16:25

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Ruth,
I can't be 100% sure 1,25-D functions as an antagonist, as we really don't know very much about the GCR (GCR-null mice do not survive gestation), or the residues which are required to activate the receptors, or whether there are multiple modes of activation.

However, there appears to be sufficient divergence between the residues which are 'contacted' and the mode of contact, to make me think that antagonism is far more likely than agonism.
 

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 Posted: Wed Jun 27th, 2007 06:12

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http://winmlm.neostrada.pl/mp/affinities/1.jpg

Interestingly all D metabolites have higher affinities to Thyroid alpha 1 and Thyroid beta 1, than they have to VDR. I thought that metabolite is named after the receptor it binds best. And from the table above it would seem that 1,25-D is more like a thyroid ligand than VDR ligand. But than it comes that it is antagonist so... It would apper it has something like emergency shut down function there.

From the table above 1,25-dihydroxy has 5 times higher affinity for Thyroid alpha 1 receptor (0.03/0.006=5) than to VDR
and 1,25-D has 15 times higher affinity to Thyroid beta 1 (0.03/0.006=15) that for VDR, right ?



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Prof Trevor Marshall
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 Posted: Wed Jun 27th, 2007 08:00

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Well, not really. Those values were obtained from Autodock. A better package, XSCORE, estimates Kd values of 8.48, 8.12, 8.41, 8.05 into the VDR, GCR, Thyroid-alpha1 and Androgen receptor.
( Ki = 10 **-Kd )

Remember that concentrations are also a factor in homologous displacement , and 1,25-D is usually very low in concentration compared with the other hormones.
 

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 Posted: Wed Jun 27th, 2007 09:01

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Wrotek,

I did a little googling for "evolution of NHR" and came up with this fascinating document in a Biochem Projects 2000 folder at Stanford -- not a pub-med doc which means it is fairly READABLE!

http://cmgm.stanford.edu/biochem218/Projects%202000/suchorolski.pdf

Just a few lines within the first pages:

"Considering that the pathways for catabolism of each ligand is relatively more complex than the single transcript necessary for each nuclear receptor,  some authors have claimed that the co-evolution of the hormonal pathway lies mostly with the receptor evolving around a set of fairly stable ligands."

(and goes on to mention many orphan receptors for which no ligands have been identified and other things).

Fascinating stuff!

jim

 

 



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wrotek
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 Posted: Wed Jun 27th, 2007 09:43

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Is this ok ? What about units ?

Last edited on Wed Jun 27th, 2007 09:44 by wrotek



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 Posted: Wed Jun 27th, 2007 09:46

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8.05 not 8.95
Ki is molar, VDR is therefore 3.31 nanomolar, for example
 

Last edited on Wed Jun 27th, 2007 10:33 by Prof Trevor Marshall

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 Posted: Wed Jun 27th, 2007 10:44

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:) OK ?
VDR                         Kd =8.48 ,   Ki=0.000000003311311215  mol  =  3.31 nmol
GCR                          Kd =8,12 ,  Ki=0.00000000758577575    mol  =  7,58 nmol
Thyroid-alpha1         Kd =8,41 ,  Ki=0,00000000389045145    mol  =  3,89 nmol
Androgen receptor.  Kd =8.05 ,  Ki=0,000000008912509381  mol  =  8.91 nmol  

So the new additional variables actually make ligands less compatible ?

Last edited on Wed Jun 27th, 2007 10:54 by wrotek



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 Posted: Fri Jun 29th, 2007 08:01

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Dr. Marshall,

Thank you for the lucid reply.  I've mulled this over many times in my mind, and it reminds me of "stress corrosion cracking"  or SCC of metals.  For the non-mettalurgical reader, "stress corrosion cracking" occurs when a major stress leads to the electrochemcial process we know as corrosion.  The corrosion is the result of an energy effect, and the chemical process runs along grain boundaries  and then a crack occurs in the metal.  Such failures can be disasterous; i.e.,  bridges collapse.  Steam turbines go down.  Engines die.

The analogy between SCC and biology runs like this:   the VDR is getting stressed with  CWD bacteria, and over time, leads to the excess D125 from the fortified food supply going to othr ligands and receptors (corrosion?) and then our bodies fail (i.e., crack). 

Stress is the driving force in SCC; CWD bacteria are the driving force in chronic disease and amplified by the unsafe food supply (i.e., foods fortified in D25).


Does the analogy work? 

Sherry


Last edited on Fri Jun 29th, 2007 09:10 by scooker48



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tickbite
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 Posted: Thu Jul 19th, 2007 05:45

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Simulations unravel outer membrane transport mechanism

http://www.life.uiuc.edu/emad/TonB-BtuB/btub-2.5Ans.mpg

Not that this specifically pertains to anything much relevant other than gram-negtive bacteria. It's just nice to see that others are practicing the same sort of molecular modelling as Dr. Marshall and in my mind at least, it lets me know Trevor isn't a quack-pot! :D LOL



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 Posted: Thu Jul 19th, 2007 08:03

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Greg,
"Any sufficiently advanced technology is indistinguishable from magic" - Arthur C. Clarke.

IngeD
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 Posted: Fri Jul 20th, 2007 11:33

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Reminds me of a german poem that my father used to quote to me:

The dove said to the eagle "beyond knowledge comes belief" and the eagle replied "yes, but where you already believe, I still know".

Flying higher the eagle had a much wider horizon. It's just such a pity that the doves don't often listen to the eagles! Inge.



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kess3881
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 Posted: Mon Aug 6th, 2007 11:56

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curious as to the doctor's thoughts on this article on science daily. The title had to do with bacteria no longer being able to hide from testing with some new device or method. Also mentioned biofilms etc... link is:

http://www.sciencedaily.com/releases/2007/08/070802095344.htm


, does this apply to the cwd bacteria in th-1 as well?

Last edited on Mon Aug 6th, 2007 11:57 by kess3881



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