X-Message-Number: 16965
From:
Date: Thu, 12 Jul 2001 04:17:09 EDT
Subject: LN2 lighting bug / fire fly results--Revisited.
Cryonet:
The primary interest in these experiments is ultimately to discover what
category of molecular processes, structures and machinery have survived the
deep freeze. (These answers might have at least some minimal implications
for us somehow.) Discoveries may include intact specialized cell functions
and some of their organelles with functioning bi-lipid membranes and
functioning internal vesicle transport mechanisms across cell membranes for
activation of this luminescent reaction. We already know that a particularly
specialized and complicated protein, luciferin, has survived the LN2 in tact
as well as its equally important enzyme partner, luciferase. In addition,
there may also be one of more "helper enzymes" and/or necessary ligands
("special regulatory key-like molecules that must be attached to their
correct receptor sites on the proteins in order for this light to be
emitted). At this point, we know that something is working that has
withstood the LN2 insult. The enzyme machinery complex has apparently being
left substantially in tact after LN2 storage. It is not out of control as it
glows steadily rather that express itself as a sudden single bright burnout
flash. I concede that there is a possibility that these observations might
possibly still exist even if host cells were very damaged. I hope to find
that out also. This approach is somewhat of the cart before the horse as
typically one would have read and understood the existing literature on the
workings of these systems and then test for verification. But this could be
a good learning exercise (for me and perhaps vicariously for you if you have
the interest now or later)--by deriving the answers rather than be given the
answers.
Later, after gathering some data and trying to form a reasonable hypothesis
or two, I will read everything I can regarding research in this area. One
thing that I almost certainly will do, just for fun, but also as a potential
publicity item for cryonics leaders if they would like it, is to demonstrate
the fact that LN2 is basically "forever." For instance, when the first fly
came out of LN2 after a little over two hours last night, what struck me as
neat was the knowledge that the same fly would have made the same exit and
presentation 2000 years from now. For this reason, I may warehouse a few
dozen flies to be "revived" every five years or so--you know, like the Third
Bi-Decade Fire Fly Revival Show held this year in beautiful Rio...buy your
tickets now and arrange for lodging early...--OK, I digress. (Didn't Geraldo
do something like that one time with someone's long lost locked safe?)
Another fire fly was retrieved from liquid nitrogen tonight. This time I
turned the lights down very low in "the lab" to see exactly when illumination
would start. First note that at room temperature the ampoules (tiny polymer
test tubes) are very slightly "frosted"/textured ("translucent" I believe may
be the right word). Immediately out of LN2 they are somewhat more so due an
immediate condensation and freezing of atmospheric water vapor on their
exteriors. However, even so, I was able to determine that there was no light
emission from the fire fly immediately upon retrieval from the dewar. I
immediately unscrewed the cap of the ampoule and dumped the insect carcass on
the lab bench. The tail end of the fire fly began illuminating within two
seconds as the body quickly warmed. It was reasonably bright but not half as
bright as a living fly. (To make a more definitive determination this time
regarding the relative brightness, I had just "ampouled" five new specimens
caught earlier this evening, and thus I could better make the comparative
judgment regarding intensity as they were also on the bench.)
The difference in illumination pattern is that the LN2 flies demonstrate a
constant glow of milder intensity while the living flies largely flash
brightly like turning a switch on and off (while they too can and do
sometimes emit a similar steady, low intensity illumination as I witnessed
tonight--in fact, the LN2 fly's glow was at least as strong as these constant
emission patterns of living bugs.)
An interesting observation was that the LN2 fly's initial constant glow ended
after approximately 1/2 minute or so to fade to almost completely "black" and
stayed "off" for approximately 1/2 minute or so. And then it just as quickly
came back up like someone briskly turning up a dimmer switch attached to an
incandescent light fixture (at which point it proceeded to glow steadily for
well over an hour to fade out completely, as last time, at about 1.5 hours).
I believe I may have witnessed a temperature-based phase change sweep over
the bug's body/lighting apparatus whereby the momentary freezing of water
molecules (perhaps from some colder crystalline order of ice, or even
vitrification?) disabled the photochemical reaction (i.e., the luminescence
may proceed at temperatures lower than ice?--now that would be interesting!)
Speaking of, these tiny insects may be instantly vitrified with their small
mass--if not, because of the air cushion in there ampoules, I might try
suspending one in a small sock for direct contact with the LN2 and see if
there is any change in luminescence revival. However, I have I have a hunch
that this particular phenomenon may be reliably reproduced with this type
experiment in the future. A high confidence, accurate *explaination* may not
come for some time. Actually it could be the depletion of one source of
energy and the initialization of another (as some of our muscle cells will
revert back and forth under some circumstances from aerobic to anaerobic
systems of energy).
Of the five fire flies I caught tonight around the apartment complex swimming
pool (by the way, having a real cute college girl in a bikini with a mixed
drink in her hand be so interested and delighted in what I was doing may have
inspired the new search tonight--I now brush my teeth and ware contact lenses
instead of glasses for more effective fire fly pouching), I put three in LN2
storage, one of the ampoules of which was full of water and fire fly. (I
first chilled the water to its freezing point and had also chilled the
selected fire fly to a near "hibernation" state before combining the two in a
new ampoule for the LN2 dewar. The remaining two ampoules went into the
freezer and one of them was similarly treated with cold water.
The reason for the water is two fold. Firstly, assuming that the initial
on/off routine will prove to be a standard "revival ritual" for the
illumination, the surrounding shell of frozen water may inspire an
alternative initial blackout period (as previously cited). It may delay it,
prevent it or not affect it. We will see. But it will be held at a
temperature no higher than that of ice for a period of time. The second
reason is to take an easy stab at determining whether "respiration" is
required (i.e., the "burning," or more accurately, the oxidizing of molecular
oxygen). If there is minimal or no glowing in the iced specimens, it may
indicate that oxygen is needed. These insects likely breath differently from
higher form beings--some insects breath from their abdomen parts and may not
require that the bug be living. If there is no glowing, we may be able to
determine that the illumination requires such an oxygen availability. Of
course, it very well could be an entirely anaerobic reaction whereby the ice
will make no difference. All of this provided that any such additional form
of energy is needed at all since there may be enough ATP stockpiled for the
90 minutes. The breakdown/utilization of ATP (Adenosine Tri-Phosphate)
itself requires no oxidant as it only involves hydrolysis (i.e., the
splitting of a water molecule and adding the parts, a hydroxide ion, OH, and
a hydrogen atom, H, to the molecule hydrolyzed).
I will keep you posted, and will make any factual corrections to the above as
I may become aware of them.
Regards,
David C. Johnson, Raleigh, NC
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