Human Frontiers has just posted a highlight of our recent Nature Methods paper in their "hot of the press" section.

www.hfsp.org

Inhibtion of leukemia in vivo using a synthetic peptide.

Walensky et al. published a wonderful paper in Science (2004) 305: 1466-70 in which they develop a technqiue to enhance the alpha-helical folding of a peptide designed to stimualte apoptosis of leukemia cells. The peptide selected is the BH3 domain from the "BID" protein. I must admit I know nothing about these specific proteins or signaling pathways but the authors marshall an array of multidisciplinary studies that are dazzling and compelling to show that organic synthesis of a BH3 analog can produce a peptide with dramatically increased alpha-helical structure. They use CD spectroscopy to show that a hydrocarbon bridge of fixed length and conformation (8 carbons with a central cis double bond) between adjacent turns of the peptide alpha helix increases the alpha-helical content from 15 to 85%. This secondary structural motif is vital for the interaction with the down-stream targets, BAX and BAK (proteins that stimulate mitochodrial driven apoptosis). Not only does this enhancement increase the potency of the peptide, but it serves two other essential functions: (1) protects the peptide against degradation in vitro and in vivo; and (2) allows the peptide to penetrate the plasma membrane to the mitochodria. The authors use confocal microscopy, FACS analysis and multidimensional NMR to show how their "peptide stapling" technique works. The most striking results are saved till last. They find the mean survival of leukimic mice is increased from 5 to 11 days (total body luminescence is used to quantify tumor size). Thus, the study is a true multidisciplinary tour-de-force, encompassing many state of the arts technqiues, leading to a therapeutically important outcome.

Proper use of DM-nitrophen.

If anyone is interested in seeing DM-nitrophen used in a quantitative study, then read the very elegant paper by Bollmann & Sakmann in Nature Neuroscience last year (2005, 8: 426-434; doi:10.1038/nm1417).

One of the Holy Grails of synaptic physiology is the quantitative correlation of presynaptic calcium concentration with postsynaptic currents. The calcium microdomains that drive secretion are too fast and small to image, so a few years ago the Neher and Sakmann groups resorted to a roundabout means to get at this using calcium uncaging from DM-nitrophen and NP-EGTA (Nature (2000) 406: 889-893; Science (2000) 289: 953-957). Both groups used the calyx of Held, as it is sufficiently large and robust to allow double patching of pre and postsynaptic cells. Global uncaging of calcium throughout the presynaptic cell produced a sustained elevation in calcium that can be measured. (I remember when I first met Gerrard Borst as a postdoc in Heidelberg, he told me he was happy if he got 3 pairs a week! I can only assume the success rate has gone up since then, as Bollmann a mind-boggling amount of data.) Bollmann filled many presynaptic cells with various proportions of DM-nitrophen, calcium, oregon green BAPTA-5N and buffer (EGTA or BAPTA). These cocktails permit different calcium concentration waveforms to be generated in a reproducible and quantitative manner by uncaging from DM-nitrophen. (A Nd:YAG laser was used for uncaging; pulse-width 3 ns.) In the great tradition of Hodgkin and Huxley, Bollmann also produced a model of the dependence of postsynaptic current upon the occupany of calcium on the secretory machinery to account for his data, and predict rise times of [calcium] vs. postsynaptic current and explain superlinear amplification during paired-pulse facilitation. A proper use of DM-nitrophen indeed!

In vivo uncaging of calcium?

The Nedergaard lab have a paper coming out soon in Nature Neuroscience (published online: 25 December 2005; | doi:10.1038/nn1623) in which they cliam to use DM-nitrophen AM for UV uncaging of calcium in living mice. The images presented are quite beautiful, but the uncaging is impossible as DM-nitrophen has to be caged magnesium, as mentioned earlier on my weblog. They have no excuse, as Molecular Probes sell the probe as "DMNB-EDTA", since Calbiochem trademarked "DM-nitrophen" (somehow). It says EDTA in the name of the compound: has everyone forgotten in their rush to do biology, that this binds magnesium very well? That's why everyone uses the calcium selective probes EGTA or BAPTA for intracellular work.

Our paper on two-photon uncaging of IP3 in living cells came out today in ChemBioChem.
DOI: 10.1002/cbic.200500345
Our new cage (NV-IP3) was quite photosensitive as we used only 5 mW for 100 ms to produce calcium puffs or waves (we were surprised by how facile the uncaging was!). This is the first exampe of localised photolysis of IP3 using near-IR light (we used a mode-locked Ti:sapphire laser at 710 nm).

Our Nature Methods papers was highlighted in Nature:

http://www.nature.com/nature/journal/v439/n7072/full/439004a.html

I know this looks incestuous, but in fact Nature don't do this that much.

Science published a “feature” on our Nature Methods paper:

http://sciencenow.sciencemag.org/cgi/content/full/2005/1223/2

DM-nitrophen AM ester is caged magnesium.

There have been several reports using DM-nitrophen to photorelease calcium inside cells by both one (UV) and 2-photon photolysis [Nature (2004) 431: 195-199; Cell Calcium (2006) 39: 65-73; Gastroenterology (2002) 122:415-27]. DM-nitrophen is a caged calcium I made as postdoc in 1988. It is based upon EDTA, so has a very high affinity for calcium (so far so good) and a moderate affinity for magnesium (Dooh!!). Thus, in a normal intracellular milieu DM-nitrophen is caged magnesium. (Modeling says it is about 95% loaded with magnesium.) Thus, the calcium-induced calcium release reported in these papers must arise from photodamage of cells (either of the SR or PM), not from uncaging of calcium. The real give away in the work from the Nathanson lab in which they “load” cells with the free acid of DM-nitrophen, then use 2-photon uncaging to elicit CICR. It is clearly impossible for a small organic molecule with 4 negative charges to penetrate the plasma membrane, else why would Roger Tsien bothered developing AM-ester loading of calcium chelators?

The reviewer’s review reviewed.

We have just published a paper in Nature Methods (2006) 3: 35-40 in which we introduce a new caging chromophore to the scientific community. Our first application of the new chromophore is to "Caged Calcium". We have synthesized a photolabile EGTA derivative, and characterized this new probe chemically and biologically (viz. affinity before and after photolysis, rate of Ca release, 2-photon cross section, quantum yield, UV uncaging in muscle fibers, and 2-photon uncaging in droplets and living cells). This is the first paper in this field with such a comprehensive and complete characterization of a new probe. The properties of the chromophore are so superior to others that I regard the work as a landmark contribution to this important field.

Wen-Hong Li wrote an interesting News and Views to accompany our recent paper. In his review he mentioned “Several caged compounds useful for two-photon uncaging applications have been developed recently(refs. 3,5-8), and in some cases, rapid release of effector molecules has been demonstrated (ref.9).”

I feel that the way Dr. Li highlights these other caging chromophores is extremely unhelpful to a general reader of a News and Views. He references five other caged compounds: his ref. 5 Bhc-glu (Proc. Natl. Acad. Sci. USA (1999) 96, 1193-1200); ref. 6 azid-1 (Biophys. J. (1999) 96: 489-499; ref. 7 MNI-glu (J. Neuroscience Meth. (2001) 112: 29-42; fef. 8 Org. Lett. (2002) 4: 3419-3422; and ref. 9 DEACM-sulfate (caged protons) Angew. Chem. (2005) 44: 1195-1198, giving the impression in this context that there are plethora of caging chromophores out there that are also “useful” for 2-photon photolysis, just like NDBF-EGTA. Li does not mention how these are useful, and if one actually looks at the references one will discover why.

Of the 3 cages that have measured 2-photon cross sections (Bhc-glu, azid-1, BQH-Ac), only one has any accompanying 2-photon biology, Bhc-glu. However, the biology reported by Roger Tsien and co-workers using Bhc-glu is “odd” in that the rise-time for the AMPA receptor is reported as 200 milliseconds, rather than the normal 100 microseconds (Note added later in 2006: Sam Wang-who was the postdoc with Denk who did the work at that time- read this entry and told me this was the integrated current over the line scan, not from point uncaging. So I misunderstood this figure in his paper. I am not sure this correction makes the data or Bhc-glu any better?). Thus, it is hard to say that any real 2-photon biology was done using Bhc-glu. Furthermore, the rate of uncaging of Bhc-glu was not discussed, because it had been measured by David Trentham and colleagues in 1993 and is slow (140/s). (We mention this is our Supplementary Table.) Significantly, there has been no report of any 2-photon biology with azid-1 (the only caged calcium in Dr. Li's list, also made by Roger Tsien and co-workers) since its introduction in 1997 as this cage releases calcium too slowly for focal photolysis (rate=500/s). The MNI-glu reference used by Li has no report of any 2-photon measurements whatsoever, and the DEACM-sulfate reports a fast uncaging rate but also has no report of any 2-photon biology. In contrast, we measure the rate of calcium uncaging from NDBF-EGTA, and report a 2-photon cross section and use 2-photon uncaging to induce localised calcium release in cardiac myocytes.

Secondly, Dr. Li states that “further improvements of …its resistance to pH interference should broaden its applications in studying calcium signaling.” We feel this statement suggests that because NDBF-EGTA is based on EGTA it will not really be useful until there is no pH dependence of the calcium affinity. If this were actually the case then the two other cages we have made based on EGTA (NP-EGTA) and EDTA (DM-nitrophen) would and could not have been used in over 200 biology papers! Thus, again Dr. Li states something that a general reader of a News and Views will find misleading.

The status of N&V in the scientific community is very high. We read them so that we can understand work outside our own expertise. Thus, the factual accuracy of a N&V is paramount. We feel that Dr. Li is still reviewing our paper (the detailed comments show he was one of the reviewers of the paper), rather than communicating the great overall strengths of our work to the general reader of Nature Methods. Obviously a Errata for a N&V is ridiculous but I feel this entry goes some way to correcting Li's review.