click on the graphic to see it full size, and with a little text caption to boot
About this Graphic, "Falling Off a Log + NADH2"
Part I. The Art.
The tree, the flowers, the way the elements of the graph kind of mimic the elements of the Earth over on it's side.
This whole thing started with the graph (the Science part, Part II, infra), and after that had been sketched a few times, the "falling off a log(arithm)" part popped into my head, whilst I was emailing a German scientist about the way he grew his bugs. He grew his yeast in a shake flask, or (if he needed a big batch of them) in a sparged carboy. I wasn't too sure that everybody had even heard the phrase "(easy as) falling off a log", especially non-native-English-speaking scientists. (What the hell, I had already lost the nonscientists with "exponential growth plotted as a logarithm yielding a straight line", anyway.)
So I got the idea of a picture of a deformed log, perhaps a log with a "logarithmic" shape. It's the shape of something growing exponentially, plotted as a logarithm vs time, where after a relatively short period of time they start to starve, nonfatally. That's the way microorganisms grow, in a shake flask.
Whatever shape I was trying to come up with, it turned out not to matter; because when I took a short break and got up and walked into the living room, the tree limb with just the shape I was looking for was staring at me right outside the window. I must have spent an hour photographing that pine tree. I could have spent two more hours, but hey, it's just a job.
Maybe I should have put the graph on its side and had the Earth standing straight up. Let me try that. This is all digital, iterate all you want. Naaah, looks dumb. You gotta go with your concept, it has a life of its own. My kid Jason pointed that out, that the shape of the oxgyen tension curve resembles the shape of the Earth, now that the earth is over on it's side about ninety degrees.
I kind of held off on being too arty about this graphic. I was about to turn the oxygen curve and the growth curve and the fluorescence curve into curvy rows of alchemical symbols, the symbols for earth air fire and water.* No water, actually; NADH fluorescence would be fire, and oxygen would be air, obviously; and what could be earthier than a culture of yeast? I'd have to ask an alchemist, if I could find one, how you could make a symbol for air+water, that would properly represent the essence of the oxygen tension. But, putting in those datapoints would have been faking the data, and I'd just as soon not thus consign myself to the fires of Hell. That's where people who fake their data are despatched to, you know. People who merely fudge, nudging a few datapoints up or down and generally neatening things up a bit, they spend their eternity in Purgatory.
Mimicking the Biblical style, in the dozen or so lines at the bottom, maybe that's arty too.** It's certainly writerly. Calling NADH a "pyridine nucleotide" is old fashioned, like saying "thee" or "ye". I could have labelled it DPNH, which as I recall stands for diphospho pyridine nucleotide. Yeah, it's an art.
* The alchemical symbols for earth air fire and water, from http://www.adh.brighton.ac.uk/schoolofdesign/MA.COURSE/LFourEle.html
are very much like the symbols scientists use for the datapoints on their graphs, little circles and squares and half-filled-in circles, and triangles, e.g. http://www.frogojt.com/yeast1fig.html
** I tried to do something with "In the Beginning", or Genesis, tying it into "genes", and "genetics", and "gene expression"; something clever and Shakespearean. Didn't work out. Maybe it will come to me later, after I do a little more research on whether genesis (and Genesis) has anything to do with genes. Probably not necessary; you just intone the phrase "In the Beginning"; your audience gets the whole picture by way of allusion and synecdoche.
Part II. The Science
click on the graphic to see it full size (links to the same graphic as the beginning of this explication)
Long story, and convoluted.
There I was, in Philadelphia in the late Sixties, having a great time playing with the experimental system Professor Chance and I had come up with. When I'd grow a culture of yeast under our defined conditions, I could do it the same way every time. And every time they would run out of fuel at about the same time, give or take a matter of minutes. You could add poisons, or other substrates, or change whatever you wanted, but the basic system was under control. As it should be. Good system.
If I were Charles Darwin, I'd spend the next paragraph or two railing against all of my colleagues who had a not-so-good system for growing bugs, mostly in a shake flask. Instead, I'll wait a few paragraphs to do that.
One of the things I chose to do with that system was to measure pyridine nucleotide fluorescence, whole cell pyridine nucleotide fluorescence, mostly due (in most cells) to the fluorescence emitted by NADH when it's in its reduced state and hit with a barrage of photons of ultraviolet light, down there in the 200 nm or so range.
Here's a quote from http://www.frogojt.com/Richard1969.html
... in this particular experiment (which was never actually published), in addition to measuring the culture's respiration, rotenone sensitivity, pH, and turbidity, I was going to measure the entire culture's pyridine nucleotide fluorescence (in response to excitation by UV light of 240 or so nanometers.) In the foreground are two gas cylinders, one of oxygen and one of nitrogen. I would gas the culture with nitrogen and measure the fluorescence, and then with oxygen, to calibrate the fluorimeter. Then I would measure and record continuously the fluorescence of the entire cylinderful of growing yeast cells, at (as I recall) a wavelength of 340 nanometers.
This turned out to be a fairly ambitious undertaking, but as I said in http://www.frogojt.com/Richard1969.html ,
... when I said to Dr Chance (Britton Chance, the Director of the Johnson Foundation for Medical Physics and Chairman of the Dept of Biophysics and Physical Biochemistry and Benjamin Franklin Professor of the University of Pennsylvania, now Emeritus) that I wanted to measure total pyridine nucleotide fluorescence, in vivo, in a soupy yeast culture, Chance didn't bat an eyelash.
I worked at it for a few days on end to make that measurement. And, against all odds (nobody had ever done anything quite like it before, it turned out, sorting out the NADH signal from a soup like that), a signal was measured reproducibly.
Quoting further from http://www.frogojt.com/Richard1969.html
So the total pyridine nucleotide fluorescence increased more or less exponentially with the increase in the number of cells. Frankly, I can't remember what relationship there was between the onset of rotenone sensitivity and pyridine nucleotide fluorescence. You see, it's not like the yeast run out of NADH, or run out of fluorescence. What I think we were measuring was How reduced are the Cells? When they would run out of ethanol (their only carbon and energy source) they (all of them, en masse) would go from being pretty much totally reduced to pretty much totally oxidized. That just blew me away. That was like finding out what Monsieur Jourdain found out from the Philosophy Master in Moliere's le Bourgeois Gentilhomme: That he'd been speaking in Prose, all his life! We are all of us, mostly, Reduced. ***
In the real world, as quoted up above, that experiment never got published. Now why was that?
Well (he said, puffing on his meerschaum), two things:
The yeast cells went from growing to non-growing, when they as usual ran out of ethanol to eat. But they didn't acquire the rotenone sensitivity that they "should have" acquired when they went into their stationary phase. That was one thing. That was pointed out to me with a withering stare by another scientist at the Johnson Foundation.
Second, there was no real experiment. I was just measuring this stuff for the hell of it. To see what it looked like. To see what was happening, in the finest tradition of gentleman science.
Now that's not to say I might not have gotten around to publishing it some day. But as time went on, I got more and more estranged from doing science, and then the day came that all of my records (notebooks, stripcharts, reprints, drafts, the works) got rained on in my neighbor's leaky garage. So I chucked the whole non-waterproof footlocker into the trash, data and all, out here in Berkeley.
But even that is a little after the fact. The fact is, that I had been talked out of publishing the results, by that other scientist at the Johnson Foundation, the one with the withering eye. That's what I puzzle about now; how I could have been so naive, or gullible, or obedient, or whatever, when my whole life I've been way too aggressive, in general. Hell, I've been a full time truckdriver in my day, and you don't get through a day of that kind of activity by being meek. Nosirreebob.
I have thought about those experiments with NADH fluorescence about twice a day ever since, a time of about thirty years or more. Lately I've even taken to kicking myself for tossing out the rain-damaged data. The only consolation is that one could, if one so desired, always set everything back up and repeat it. This is, after all, Science. Probably the technology has improved a little bit, and it might even be easier for that. Less cumbersome, anyway.
One explanation that has me just fascinated is that the UV radiation just knocked the hell out of whatever was supposed to get those yeast ready for their stationary phase caused by running out of fuel. Now that would have been a result worth publishing in any journal.
But the other thing I've thought of, several times a day, is that the results were obtained with sufficient oxygen for the yeast cells. Most microbiologists don't pay any attention to that. (This is the Darwinian carping, Part I supra.) They grow their cells in a flask and shake them, in a shake flask; or they bubble with air (they sparge them, with air; that little sintered glass job that makes the bubbles is a sparger.) Their oxygen supply is insufficient.**** That's what the oxygen concentration curve on the "Falling Off A Log + NADH" graphic is all about. Their oxygen tension (dissolved oxygen concentration) is zero or thereabouts. As far as their oxygen supply goes, they're runnin' on empty, and it's getting emptier all the time. Falling off the log, you might say.
What's that supposed to mean? I'm right, they're all wrong? That the shake flasks, the canonical shake flask, constitutes a systematic error? That all these gene expression scientists, for example, are doing something wrong at step one? Because what this is really all about is that some gene expression scientist is supposed to take a look, someday, at microarrays of Candida utilis as they go through a cycle or two of the Acquisition and Loss of Rotenone Sensitivity, through a well defined stationary phase and back again. You just might find that clutch, but not if you don't grow the cells right; not if you grow your cells in a shake flask.
Yeah, maybe so. This arty-looking piece here, with the tree on its side, is supposed to be a rendering of the concept, that if you run out of oxygen, and your whole system changes as the redox potential of the system goes from reduced to oxidized, you've got a lifechanging situation, something God really gets involved with; something that would surely affect gene expression.
Here's my problem: I never did the experiment that's depicted in the graph. I never actually grew a culture of cells, and measured the NADH fluorescence when they slowly ran out of oxygen, but kept on going and growing, albeit slower and slower. As I recall. Maybe I did, and just don't recall it. I feel like I measured that, that I just turned off the air supply (the oxygen supply, actually) and if I had those damned stripcharts handy, I'd be able to piece it together. Rain or no rain. Forensics.
What I do recall, is that when the little guys ran out of food (ethanol, an oxidizable substrate and the source of their energy) they would get pretty oxidized, i.e. a lot less fluorescent. What I vaguely remember is that when they would run out of oxygen, they would get a lot more oxidized also. And now that I think about it, that's pretty oxymoronic. Oxy_moronic. Run out of oxygen, get more oxidized. That's how I remember it, paradoxical as it seems when you stop to think about it.
Maybe it's that FLOW of electrons that gets the NADH in the hyper state of being really really reduced. What I remember pretty clearly is that when the culture was growing at a fast clip, generation times of only about an hour or so, as I recall, the fluorescence was obviously above the fluorescence of the calibration for that sort of thing, i.e. what you'd get when you gassed with nitrogen.
One thing for sure: Whatever you do, you better not use a shake flask to grow the yeast, or any other bug you've got to culture. And sparging with air doesn't cut it either.
*** Maybe that's drawing too broad an inference from the experiment. Our yeast, running full bore, were certainly mostly reduced. I wonder about us. We are a collection of many different kinds of eukaryotic cells, and they're growing or not growing fast or not so fast, depending on their station in life. Maybe they have the bioenergetic equivalent of a clutch.
**** When I point that out, about the shake flask or about the aeration with a sparger, e.g. in the emails I have elected not to append to this footnote, the usual scientist's rejoinder is "It doesn't matter." That's different than their saying "You're wrong." It's more like they're saying, "I hear you. Now fuck off." Science is definitely like that.
(c) 2002 Richard Katz. FrogOJT Systems, Berkeley, CA. email to Richard Katz
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