Oliver Smithies:

[00:00:00] So this is book capital R’, September 5th, 2004 running through May 4th, 2005.  Still thinking about the kidney model at the beginning page, Sunday, September 5th, thoughts on the kidney model.  Likely that the energy of the loop of Henle comes from the thick descending lymph cells.  And for the collecting duct energy comes from the thick ascending lymph.  In other words you generate the energy in the thick parts of the nephron and use it in the thin parts to move salt and the like.  I don’t think a novel idea.  But anyway quite definite that energy is put in [00:01:00] in the thick tubule cells where lots of mitochondria and then it’s used by switching channels in the thin parts which don’t have many mitochondria.  And so what can the tubular fluid carry to deliver the energy without needing mitochondria?  ATP, creatine phosphate, acyl-CoA, ionic balances.  In fact of course it’s sodium.  Look carefully at water handling in diabetes mellitus versus more obvious way it’s handled in diabetes insipidus.  Why does high glucose cause, or does it, a high volume?  Or is it the inability of the tubule cells to intake and utilize glucose as an energy source?  [00:02:00] Water, urea, and sodium chloride are quite enough to begin with.

My view not very long after that was clearly that changes in salt concentration are used and sodium concentration are used to pump other substances, with a countercurrent helping to concentrate sodium chloride in the extracellular fluid.  More constructs being talked about the following pages.  Usual sort of thing.  [00:03:00] Dupdel, HPRT being worked at still.  Some thoughts Wednesday, 29th, page 19.  That Sandy’s equation f equals e to the minus pi (R plus r) squared n has the same form as the Poisson distribution where lambda would be equal to np in normal distribution.  So pi (R plus r) squared is [00:04:00] in a sense p.  And the n fibers in the sense of the number of trials.  I realize that that’s close to what is in my head these days and that is that the equation has square terms in it.  In other words it’s a series because if you calculate the available space or the excluded space with a dilute gel then that’s very close to pi (R plus r) squared times 1.  But when you have more concentrated gel [00:05:00] some areas get counted — some parts of the excluded areas are counted twice.  So you have to have a correction.  So that’s why there’s an addition of a pi (R squared squared) term, etc.

So here were some tests on page 21, 24th of September.  One-day-old long BGIIAC pups.  That’s with the insulators.  The adult shows about zero green nuclei in the ventricles.  In other words it’s switched off. However [00:06:00] here is the first crude picture.  It’s working well.  It’s working as expected.  One-day neonatal heart ventricle, but better pictures are on the way.

Still thinking about Howard Rockman’s hypertrophy experiment.  So it [00:07:00] allowed me to conclude that alpha-myosin beta-myosin heavy chain — alpha-myosin heavy chain, beta-myosin heavy chain, collagen I, ANP, and BNP are affected by TAC by the transaortic coarctation whereas many more things are altered by running, alpha-myosin, beta-myosin heavy chain genes, MCAD, ATR1AAAM, NPRA and ET1 and AGT exon 3.  So with a result that run causes general increases but TAC causes some decreases.  [00:08:00] Pa pa pa switching, exclamation mark.  Confirms that alpha-myosin heavy chain and beta-myosin heavy chain are good choices.  But I need to recalculate those correlations that involve synthetic numbers.  They are using only the real data because I put in some unknown numbers in these simulations.  I found a publication that other people were thinking of a toggle switch underlying vascular disease, page 29.  [00:09:00] Had a visit from Hunter Scott for a day to have some instruction in Stella modeling and attempted his particular model.  Was somewhat successful.  Some success.  Continuing to make plasmids.  But on [00:10:00] Friday, December 10th on mRNA versus GFP fixation and whether paraformaldehyde can be used.  It preserves GFP but loses the RNA and ethanol preserves the RNA but loses GFP.  And so various epoxy butane might be a good cross linker.  Chuck Carlin had used it. [00:11:00] Wednesday, December 22nd, page 73.  Joe noticed a female about, an [00:12:00] eight-month-old, close to death with A mite of C AN in the ANF locus and sacrificed it with the heart on the left.  So very hypertrophic.  With a possibility that mite of cyan in the atria causes mitochondrial disruption and death, model of progressive heart failure due to atrial [00:13:00] fibrillation and clotting was the thought.  But the heart shows great thickening of the atrium.  Exciting at the time.  Transferred some of my Dupdel plasmids to Open Biosystems so they could make those available to other people, Wednesday, January 12th, page 79.  [00:14:00] Continuing with making plasmids.  One last time again Friday, February 4th, page 95.  Try for the last time in Dupdel to put in a fragment.  Of course it wasn’t the last time.  The ligation [00:15:00] was repeated on the next page.  Continuing with these various plasmids.  Getting I think a little lost really.  [00:16:00]

New promoter.  CY promoter.  Driving mitochondrial cyan fluorescent gene.  Saturday, March 5th, page 115.  Now a little beginning of work on the preeclampsia type situation page 125, Monday, March 14th.  Soluble slit.  [00:17:00] Vicky Bausch gave us a mouse soluble slit full-length clone.  And thinking about putting that into the HPRT locus so as to control S slit without having to use an adenovirus.  The idea being to drive it with an AGT promoter.  [00:18:00] Or the CYP1 promoter.  Main idea being to get soluble slit 1 under our control without adenovirus.  So here on page 129 Thursday, March 24th an entry with S flit highlighted.  So got plasmid R’129 full length of S flit [00:19:00] coding sequence.  So this idea being pursued on page 133 on Tuesday, March 29th.  S flit coding sequences.  With nine steps to get it into CYP-driven situation.  [00:20:00] And step seven was on page 137 Monday, April 4th, Nobuyo’s birthday.  [00:21:00] The insulators for Dominic Ciavatta were sequenced and found useful — on construct were found useful on page 141 Thursday, April 7th.  pDC184.  In two orientations and blue and red.  So that was now available.  Working on step three of an S flit construct [00:22:00] page 147 Tuesday, April 19th.  And revise plan on page 153 Saturday, April 23rd.  Now running to 12 steps.  [00:23:00] Some thoughts on myosin heavy chain controls Wednesday, April 27th page 157, with about six steps.  And then six comments and then comment seven.  Why worry?  Beta YMHC is working at the beta-MHC locus and alpha cyan is — beta yellow MHC is working at the beta-myosin heavy chain locus.  And alpha cyan is working at the HPRT locus so why worry?  And the book ends Sunday, May 8th by looking again at sequences [00:24:00] of the blue colonies in about 26 number 13 ending book R’.