Oliver Smithies:

[00:00:00] So this is book capital Q’ beginning January 13th, 2004 nine years ago, thereabouts.  No, let’s see, 11 years ago.  And ending September 2nd, 2004.  So it begins as usual with continuing of constructs.  Tuesday, January 13th, page three.  Some more thoughts on Ogston’s equations.  [00:01:00] Thinking about the statement about flow versus diffusion.  A more accurate statement is that as the molecular size decreases and the diffusion coefficient increases the time to reach diffusion equilibrium across the membrane will be shorter than the transmembrane flow time.  And so plasma glomerular fluid concentrations will become equal, etc.  So difficulties of what happens during equilibrium.  [00:02:00] And reaching equilibrium not immediately simple.  Making clones still.  Big part of my work.  Kanamycin procedure to get kanamycin resistance.  [00:03:00] Monday, February 23rd, page 45, a one-step assembly failed.  One construct.  But the first of a two-step assembly worked.  Getting cyan into Dominic Ciavatta’s construct.  [00:04:00] And so it goes on pages, assembly of fragments.  Positive control.  Final assemblies.  And then an interlude on Wednesday, March 10th, page 69.  Thinking about mouse retinograms.  Probably a result of talking to Simon John, who was thinking a great deal about ANF and glaucoma.  Current attempts at good imaging have been largely unsuccessful and so tried to get some images.  [00:05:00] Not very successfully.  As exemplified by March 11th, Thursday, page 71 mouse eye test.  Frustrating attempt.  But one depth of focus theory with about Super 66, etc.  Looked as if one could see blood vessels.  And trying to capture these images.  [00:06:00] How long it takes.  An example of that on Monday, March 15th, page 77.  More bulk attempt.  Four months from the start to getting here.  Still thinking about photographing the fundus.  Saturday, March 20th, page 79 and the following pages.  [00:07:00] More attempts.  Monday, April 12th, page 83, a summary of the attempts with the fundus lenses.  Not very convincing.  So new thoughts and plans.  [00:08:00] Tuesday, April 13th, page 85.  Testing the kidney hypothesis.  Nothing very striking on this page.  [00:09:00]

So some conclusions Thursday, May 20th, page 87 on retinograms.  After many more attempts we tested a Heine indirect mounted ophthalmoscope and obtained video-captured images.  Mary Elizabeth Hartnett, [00:10:00] a professor of ophthalmology, came and helped by looking with her indirect method ophthalmoscope and we then looked into the eyes of 12-month-old Akita plus minus diabetic mice with a bradykinin receptor knockout.  Found nothing remarkable.  But we did see blood vessels.  Decision being then to send fixed eyes to those who are experts in retinal studies.  And if you can’t see them, Tim Kern being the person, if he can’t see them, abandon.  If he can, reconsider.  [00:11:00] And then the fourth possibility being to attempt to modify NM100 to take fluorescent retinograms.

More urine experiments.  Thursday, April 15th, page 89.  Using different stain.  Thinking about what is the best gel to use for looking at urinary proteins.  On Wednesday, April 21st, page 93 some thoughts on stacking, etc.  [00:12:00] Standard Laemmli gel and system which I tried from page 89.  But at the bottom is a comment which is really a relevant one.  The precast 8-to-16-percent Tris-hydrochloride gel that Nobuyuki used is good.  So I’m beginning to reach the conclusion which eventually I adopted that it is silly to try to improve on the stacking gels made by commercial companies and particularly by Bio-Rad.  [00:13:00] Testing the fluorescent dyes.  SYPRO Ruby, etc.  On page 95, Wednesday, April 21st.  With SDS gel.  Regular SDS gel.  Wednesday, April 21st, page 97.

Trying to assess what to use.  [00:14:00] More thoughts on staining, April 23rd, page 101, Friday.  Sample buffer tests.  Tris-borate Monday, April 26th, with a comment on the left-hand page.  Very poor results.  Better rethink.  Slowly slowly getting there.  Tests of an outdated 4-to-15-percent Tris-hydrochloride gel.  Tris-hydrochloride with a glycine external buffer.  Tuesday, April 27th.  Glycine [00:15:00] stacking is very effective.  The sample loading buffers checked here are approximately equivalent to standard pH 6.8, etc.  Good nice result.  More tests of gels.  Simple Laemmli system.  Page 111, Wednesday, May 5th.  Bio-Rad gel 4-to-15-percent Tris-HCl.  Comparing staining with [00:16:00] SYPRO Ruby or Coomassie blue didn’t show all that much difference.  SYPRO Ruby is more trouble.

Going back to diabetic nephropathy now.  Friday, June 4th after a vacation in England.  So from May 6th to June 4th a break.  Paper rejected on bradykinin [00:17:00] action but will revise and send it to PNAS.  So this is experiments with bradykinin receptor knockout and its effects on albuminuria.

So some questions about the accumulation of proteins during — in the bradykinin receptor knockout mice.  [00:18:00] So May 18th, page 117, Charles Jennette was concerned that the Akita diabetogenic mutation in insulin 2 gene causes IgA nephropathy and that bradykinin B2 receptor absence might increase this and so account for the albuminuria.  He checked.  Therefore checked various mice for immune deposits and find that there is IgG, IgA, and IgM present in the Akita mice in the glomerulus.  But it didn’t make any difference when they were also bradykinin receptor-negative.  [00:19:00] Beginning to think about how albumin might be recovered in the kidney a little more on page 121, Tuesday, June 22nd on albumin recovery.  Recent work has identified a beta-2 microglobulin-containing neonatal Fc receptor as being present on podocytes and very intensely on the brush border and that it can exocytose IgG and albumin across cells intact.  This is working.  And my sense is it likely returns albumin to the lymphatic system.  [00:20:00] Should be able to test some aspects of this with beta-2 microglobulin minus minus which we had made, naturally have about one-tenth and about one-third normal albumin.  I think that that statement means that naturally have about one-tenth the level of the Fc receptors and have albumin at about a third normal.  Thinking about this idea, whether it might be true.  [00:21:00] One of the simplest ways will be to cross-transplant the kidneys.  Low albumin and IgG kidney into a wild type mouse.  And a normal albumin and IgG into a mouse lacking the Fc receptor.  And so that idea is detailed a little more on page 122.  [00:22:00] An FcR minus minus exchange with an FcR plus plus kidney exchange we were planning.  And continuing work on the switching pairs with MCAD and the like on page 127, Wednesday, July 7th.  Switching pairs with a little table there.  Beta-MHC switching.  MCAD switching.  Long beta G [00:23:00] switching and so on.

On Tuesday, July 27th, Wednesday, July 28th, page 141 I report having looked at Howard Rockman’s data on hypertrophy.  A paper that he had sent me.  [00:24:00] On page 141, Tuesday, July 27th, Wednesday, July 28th, a rather complex set of comparisons of existing data and correlations was attempted with not any very striking conclusion.  [00:25:00] Although considerable effort.  And this was continued on page 147, Thursday, July 29th through Tuesday, August 3rd.   More analysis of Howard Rockman’s hypertrophy considerations.  But no new correlations were found with the new variables.  [00:26:00] Back with albumin recovery on Thursday, August 6th, page 151.  Talking to Masao we concluded that the FcRn receptor fetal receptor fetal heavy chain immunoglobulin heavy chain Fc receptor probably works in the earliest proximal tubule, etc.  [00:27:00] Still working on the ANF-targeting vector.  Wednesday, August 25th, page 160 or thereabouts.  No, 157.  With the usual order of doing things and a possible error and cautions.  And so the book ends on page 163.  Insert for OSDupDel HPRT.  [00:28:00] On there trying with a comment.  Back from a Pioneer Award, Olympics you might say.  Thursday, September 2nd.  That was on invitation, an attempt to have what were called Pioneer Awards, and I got into the final round and had to go to Washington to present my ideas, which were really trying to work out Stella type programs that could be used interactively by physicians.  I didn’t get funded and in the end was rather glad because it was very hard work with the computer programming in my hands.  [00:29:00] So the book ends with still trying to make a construct, Thursday, September 2nd.