Jazz Lab Band under the direction of Steve Wright just a couple of items before our next talk here I haven't mentioned this, but sitting up in front On to the right of me here Is a display of actual Nobel Prize medals? so if you would like to see them we have them sitting over here and For the benefit of those of you who visit us regularly I'd like you to get out your calendars Next year the Nobel conference. I believe will be on October 1st and 2nd The topic is the nature of nurture. We're going to talk about issues in child development And we're very pleased that so far Jerome Kagan from Harvard will be with us to talk about temperament Robert Coleman will be with us to talk about behavior genetics Eleanor Maccabee will be with us to talk about the role of parenting And of course we need somebody to talk about the brains of developing children And we just learned last week that one of our all-time favorite speakers at this conference Eric Candell Who was just awarded the Nobel Prize for Physiology and Medicine last December has agreed to come back and to talk about the Developing brains of children with us. So we're looking forward to a very exciting conference next year as well At this time I'd like to introduce my colleague Associate professor of biology dr. Colleen Jax who will introduce dr. Prusner in 1972 Stanley Prusner began a residency in neurology at the University of California, San Francisco He had returned from a fellowship at NIH where he honed his research skills to the site of his medical internship Believing the clinical study of both normal and malfunctioning nervous systems would provide the best foundation to develop a research career in neurobiology Soon after his return to UCSF He admitted a patient dying of Kreuzfeld-Yakup disease or CJD one of several transmissible spongiform encephalothepies or TSE diseases Found both in humans and other animals TSEs were believed due to the action of a slow virus an Infectious agent that caused disease so slowly an organism might be infected for years before showing any symptoms So began dr. Prusner's pursuit to isolate and characterize this type of infectious agent his studies led him to an unorthodox explanation in 1982 he reported in the journal science That the sheep TSE Scrapey was caused by an agent without genetic information That is genetic information in the form of DNA or RNA Instead the agent was a protonaceous infectious particle that he dubbed a prion Further work by dr. Prusner and his colleagues Demonstrated that the disease-causing prion protein was an abnormally folded version of a normal protein encoded by the individual's own DNA His ideas regarded as heretical Dr. Prusner spent well over a decade convincing others of his novel explanation His status as the trailblazer in this area led to numerous awards Including election to the National Academy of Sciences and the Royal Society of London and Also the award awarding of the Albert Lasker Award for basic medical research culminating in his award of the 1997 Nobel Prize in physiology or medicine Dr. Prusner has spent his entire academic career on the faculty at UCSF The prion story continues to unfold in his lab there including the search for drugs that may be effective against prion diseases as One virologist was quoted as saying following the announcement of dr. Prusner's prize There's another Nobel Prize somewhere in this field Please greet dr. Prusner who will address the current status of his field and its future via satellite in his talk Mad cows demented people and the biology of prions So anytime you're ready dr. Prusner. Thank you for that very kind and generous introduction I hope that you can hear me and I presume that the slides now are being shown. I Want to speak today at this wonderful conference about the prion diseases and Tell you a little bit about the basic biology what we know and don't know and then to speak to you more about mad cow disease and the transmission to humans after that I would like to speak about Therapeutic approaches both with antibodies and what they reveal and then with some old drugs which are finding new uses we hope the diseases that I'll speak about today I Include Creutzfeldt-Yakob disease as was mentioned in the very kind introduction Gershman-Streusler-Schanker disease fatal insomnia scrapie of sheep bovine spongiform encephalopathy of cattle and chronic wasting disease of mule deer and elk now for many years as you heard in The introduction scrapie was thought to be caused by a virus and That's because the disease is transmissible the agent is small and filterable There's a rise in scrapie agent tighter that precedes disease and there are many strains of scrapie agent that produce different patterns of disease On this slide. I tried to outline some of the history It really begins with attempts to purify the infectivity, but those attempts Really were very very difficult Because the incubation time assay had not been developed We've gotten ahead of ourselves here But once I was able to develop a new assay which accelerated our studies nearly a hundred fold Following the leads of Richard Marsh and Richard Kimberlin who popularized the use of hamsters over mice in the late middle 1970s we were able to develop a purification scheme as shown in the middle of this slide and with that We were able to purify the infectivity sufficiently to characterize it and also to eventually discover the prion protein Which is shown in the third panel of the slide brought to the very right now following the lead of Tigva Alper and her colleagues we looked for a nucleic acid, but every time we took the purified preparations and used those preparations to Look for a nucleic acid. We were unable to find any evidence for one but when we approached this looking for a protein we found that The infectivity was reduced by procedures that modify proteins so these results argued very clearly at least in my mind that The pathogen that we were studying causing scrapie Was not a virus or a Viroid Now the reaction of the scientific community in this slide is shown by the long arm and of course That's me with brown hair at the time And these are the people with a long arm the sharks Who didn't believe all of this and rightfully so they needed to be skeptical But with a lot of data that came not only from my own work But that of many other people around the world it became very clear that prions are unique pathogens Because they do not have a DNA or RNA genome and yet they are infectious. They multiply and they contain only protein Now once we just isolated the prion protein that I showed you earlier in that cartoon we were able to determine the n-terminal sequence as shown in this slide and That was with the help of Leroy Hood and his colleagues at Caltech and then we did reverse genetics in a collaborative study with Charles Weisman who was in Zurich at the time and Eventually were able to work out the PRP gene structure as shown here The PRP gene encodes a protein of about 250 amino acids and They're both at the end and at the C-termini Small peptides are removed to produce a protein eventually of 209 amino acids Now we all have the protein PRPC shown in green But what happens in the disease is that the green bar is converted into the pink bar and That bar becomes protease resistant and when we add proteases we form a protein Which is n-terminally truncated of about 142 amino acids that we call PRP 2730 Now we don't know the detailed structure of PRP 2730 But I'm going to give you some data which gives which shows the latest information that we have about it We probably know much more about PRPC and that data comes from studies of recombinant PRP made in bacteria and Then the structure determined by NMR spectroscopy The first big fragment was done by Kurt Wutrich and his colleagues in Zurich And the one shown here was done by Tom James with the help of Peter Wright and Jane Dyson and many others and At the left-hand side you see recombinant PRP Composed of three helices and two small beta strands that are shown in the blue This approximates very closely PRPC Isolated from brain at least based on optical spectroscopy and that data is shown at the bottom where we have a large amount of alpha Helix and very little beta sheet The big change occurs when PRP scrappy is formed We see a model to the right that was developed by Fred Cohn and his colleagues and What happens based upon the optical spectroscopic work is we now have a very large amount of beta sheet So we believe that helix a disappears based upon antibody studies and We believe that much of the coil that is n-terminal to helix a All turns along with helix a turns into beta sheet Now we've began to we've begun recently to get some data to refine this model Unfortunately, we are unable to use NMR spectroscopy because PRP scrappy can only be made in animals in large quantities and furthermore It is insoluble So we don't know we need we need to be soluble to do solution NMR or To hope to hope to have any chance of crystallizing it So what we've gone to are these two-dimensional crystals that form from the n-termily truncated protein PRP 2730 When it folds up and then polymerizes into the amyloid rods And these rods at the end sometimes display the two-dimensional crystals that are shown here now if one takes pictures of these crystals and Then does image processing as shown in the various panels on this slide And then compares the protein that we looked at in the last slide With a protein that is missing 36 amino acids in the center of it, but still can turn into a prion Then we can begin to look at the differences and in the lower right-hand panel One sees these differences Which are shown in the red? So one can now begin to use this and ask and know exactly where the 36 amino acids that have been deleted are They're right in the center where those red images are and One can begin to build new models and here you see two new models One of them where we have six of the proteins that fit into this hex hexagonal array and the other is Three that's shown in panel C now We don't know whether it's three or six and we're not even sure about this model, but fortunately we have some Brand new equipment, which has just been installed Meaning that we have a very high resolution electron microscope in which we hope to get better and better data that will constrain these models Now what you see in the next slide is The model of recombinant PRP that I showed you before based upon NMR spectroscopy So we believe that that data is very very good and Now a new model of PRP scrappy instead of seeing those beta strands We see this this red helix, which is really what is called a beta helix That's over to the right and we believe that this beta helix Is the region that is producing all of the beta sheet signal when we look by optical Spectroscopy, but let me stress again. This is only a model and it may not be correct Now as PRPC is converted into PRP scrappy there are several intermediates along the way One of them we call PRP star and we believe that's when PRP is binding to a protein That we call protein X because we still don't know what it is PRP star is then converted into a protease-sensitive form of PRP scrappy discovered by Yuri Safar when he developed a new Immuno assay and then eventually this this protease-sensitive form turns into a protease-resistant form and Sometimes the cells of the brain Liberate proteases that interminably truncate the protease-resistant PRP scrappy to form PRP 2730 or slightly smaller pieces of PRP that Then polymerize into amyloid, but that let me stress is a non-obligatory event Here we see PRPC being made in the endoplasmic Particulum right next to the nucleus and then the protein makes its way through the Golgi and out to the cell surface Shown by the little green balls It's attached to the cell surface by a glycolipid anchor which can be released by the enzyme PI PLC phospholipase We believe that PRPC is converted into PRP scrappy the little squares Within these cholesterol-rich microdomains or rafts and sometimes the rafts coalesce to form cavioli If the protein is directed to clathrin-coated piths Then it is not converted into PRP scrappy and we believe that's because you need the cholesterol-rich microdomain as well as a protein that I mentioned in the previous slide protein X which is shown in pink Now eventually the PRP scrappy makes its way all the way to the bottom of the slide to the lysosomes And I'm going to come back to that a little later in the talk and tell you about how PRP scrappy is turned over now PRP We thought was a Protein which was totally unique and there was nothing else like it for a long long time. This was the conventional wisdom But in the series of studies That were carried out in collaboration with Lee Hood who was at the University of Washington at the time These were done and is now in the Institute in Seattle Along with David Westaway who's now in Toronto Richard Moore who's returned to Edinburgh and Patrick Trumblay and many others Fred Cone is still at UCSF We were able to identify a second gene and this gene is called Doppel and that's shown at the bottom Doppel contains three helices A, B and C just like PRP. It contains two dose disulfide bonds It has inlink carbohydrates. It's it anchored to the cell surface and It has peptides at each end shown in blue like PRP that are cut off when the mature protein is formed The structure of Doppel was worked out by Peter Wright and Jane Dyson and their postdoctoral fellow whopping Moe And that's shown in the left in this slide And you see that the NMR structure of Doppel is very similar to that of mouse PRP and here the first mouse PRP molecule Is shown and that structure as I mentioned before was performed by Kurt Wutrich and his colleagues in Zurich You see the two disulfide bonds in Doppel and the single disulfide bond in PRP linking the long C-terminal helices together and helix A in both cases is out to the left So these proteins are very similar in structure, but I want to stress that they're very different in sequence They diverged long long ago. This is a very ancient gene duplication And so in terms of sequence homology, they're only 25 percent homologous, but structurally they're very very homologous Now the way Doppel was discovered was that there were knockout mice that developed disease in Nagasaki, Japan These are PRP knockout mice after About a year and a half of age and these Contrasted with the knockout mice made by Charles Weissman in Zurich and the ones made by Gene Manson in Edinburgh It eventually became clear that when the then in certain constructs like the one in Nagasaki There was a high level of Doppel message and that was created by aberrant splicing that created these intergenic transcripts at very high levels Now the intergenic transcripts are there at very low levels normally So these are this is a very interesting splicing situation and three of them are normally made But they are upregulated in some PRP knockout mice and the presence of Doppel in the brain Causes neurodegeneration and the animals die, but here Doppel is the toxic molecule. It's totally different from the prion Now let us turn to the human prion diseases The sporadic form of human prion disease is the most frequent. It's found About eighty five percent it accounts for eighty five percent of all prion diseases And it's found at one per million across the earth the next most common are the inherited forms such as GSS and fatal familial insomnia. These are autosomal dominant diseases affecting fifty percent of the family members Infectious forms of the disease are Kuru Iatrogenic Croix-Fell-Yakub disease caused by growth hormone derived from human pituitaries before 1985 when recombinant growth hormone Became the standard of medical care and more recently new variant CJD that we'll talk about sporadic CJD is a disease of older people and if you look at the solid black line you see that The death rates are maximal for sporadic CJD between the ages of 70 and 80 So this is a disease of older people It's an age-dependent disease and we don't understand why age is so important But like Alzheimer's disease like Parkinson's disease like ALS. This is an age-dependent neurodegenerative disease Now in terms of the genetic forms of the disease the first pedigree was drawn as early as 1930 but no one understood the significance of this in 1973 Ray Roos who's currently the head of neurology at the University of Chicago who was working with Carlton Gadgetseck and Joe Gibbs at the NIH at the time published a paper on the transmission of familial CJD cases the monkeys and apes The re-explanations were offered over the over the Subsequent ten years to explain this work first that the CJD virus was transmitted among family members living in close proximity Second there was a genetic predisposition to a ubiquitous CJD virus or third like AIDS There was vertical transmission of the CJD virus from parent offspring all of these turned out to be incorrect and In 1989 Karen Chow who was a neurology resident who had completed a residency I should say at UCSF and then came to work as a postdoctoral fellow with me Discovered that there was a mutation in the PRP gene that was causing familial forms of prion disease Karen is now a professor of neurology at the University of Minnesota in Minneapolis very close to you What Karen did was to clarify this entire area of biology and her experiments were absolutely critical in The development of the prion story it became clear from her work in a collaborative study with Tim Crow in England and with your got it Columbia University in New York that Mutant PRPC was Refolding more readily into PRP scrappy when a mutation was present that caused a familial form of these diseases Now let us turn to the mad cows in Europe for a moment The transmission of prions from mad cows to people in Europe has created a public health crisis That threatens the food and blood supplies worldwide and many of you probably realized that about a month ago the first case of mad cow disease was discovered in Japan and In recent weeks the scare from this one case of mad cow disease has decreased the meat consumption in Japan by 20 to 30 percent Now how did mad cow disease arise mad cow disease is due to industrial cannibalism and we believe that it arose either from sheep shown at the top right with passage into cows or it began as a spot as a sporadic case of Pryon disease in a cow as shown at the bottom left whichever way it began mad cow disease then spread To humans from the consumption of beef now I'm going to show you some data as long and talk about other people's work at the same time Which really makes a very tight link between mad cows and the development of new variants CJD This is one of many many newspaper articles with more than Describing the horrors of a hundred young people who have now died of this horrible of this awful disease So these are primarily teenagers and young adults with very few people over the age of 30 The top pathology is very atypical as I'll show you in a moment the disease appeared about A decade after The cows if you look at the small graph you see the peak in 1992 of cattle with BSE the Brown squares and the first patients With the disease shown in 1995 and the numbers going up year by year the disease Is restricted to the UK except for two cases and three cases in France and one in Ireland and We'll talk more about the link between mad cow disease and BSE in a moment This is an MRI scan of a patient who is dying of new variants CJD this is a t2 weighted image and You can see the very intense White areas and then to the to the right and the left of these white areas in the center of the brain We see more intensities and the problem with this slide I guess is that I really can't point this out because I've lost the use of the cursor in the power book So maybe the best thing to do is say that this is a very similar picture to That found in sporadic CJD, but at the bottom you see these sort of hazy white images They're very difficult though for me to describe without the cursor and But this is a very typical image of a patient dying of new variants CJD and it differs from sporadic CJD Now the pathology Pathology is shown here and you see the cortex of the brain and you see the cerebellum and there are all these vacuoles which are found and These vacuoles have plaques within them and this is very typical of new variants CJD and Then the presence of plaques is different In sporadic CJD where there are no vacuoles surrounding the plaques Now how do we try to link? New variants CJD with cattle or to show that it is unrelated What's happened in the in the last few years because it has become very clear that the big Biological question which made people think that a virus causes these diseases has been answered and that Question was how can there be different strains of prions or distinct varieties of prions? turns out that the distinct Varieties of prions are enciphered in the confirmation of the prion protein PRP scrappy and not in a nucleic acid. This makes prions much much different from viruses of course and We never thought that distinct phenotypes could be enciphered in a protein, but that's what happens Now how can we look at the phenotype of the disease? We can measure incubation times which I'll show you we can look at evacuation profiles and We can begin to look at the molecular properties of PRP scrappy such as the size on an electrophoretic gel These are experiments by Mike Scott where we took New variants CJD and We passages it into mice that are susceptible to human prions and you see that if we put sporadic CJD Into these mice we have incubation times of about 200 days But with new variants CJD the incubation time is over 500 days and these mice are not susceptible to prions from mad cows If we now look at this slide You see the curve all the way out to the right the black diamonds that shows you that Animals get sick between three hundred and seven hundred days on the initial passage into these mice carrying the human PRP gene or a chimeric human mouse PRP gene on second passage the white squares the Incubation times fall into two groups, so there's a shoulder in the curve So one group is about 200 days the others about 350 days on third passage We now see the the red and The blue curves so the blue curve represents the long incubation time and the Red curve the short one, so we have at least two strains that we have been able to Bring out and all the data to the right, which is a little too complicated to go through Shows that they're in fact many more strains Now we got a completely different picture when Mike Scott and Glenn Telling and Jim Mastriani and others And Jim Mastriani is now at the University of Chicago and Glenn Telling at the University of Kentucky When we carried out a series of experiments Where we passage new variants CJD into transgenic mice that express a bovine PRP gene Here you see that all the mice become ill at 260 days and sporadic and familial CJD do not make these mice sick About the same incubation time is required for BSE and this end of shorter time for scraping Here's the actual data simply showing BSE Into mad cat it from mad cows 240 days and on second passage in these bovine eyes mice it's 236 days so the curves overlie each other That's slightly different for new variants CJD here. We have human prions But now in contrast to the human eyes mice here with a bovine eyes mice all of them get sick with an incubation time of about 270 days and on next passage There's a 45-day reduction So this would be called by many people a species barrier And then on subsequent passage it's the same here you see neuropathology and This is very good evidence that BSE is The cause of new variants each AD this is the work of Steve D. Arman when sheep scrapie Was passages Into these bovine eyes mice We see no amyloid plaques the dark staining represents the amyloid and in the ponds We only see a fine granular appearance, but in the corpus closer than the ponds of bovine eyes mice Inoculated with BSE from cows or new variants CJD from humans We see an indistinguishable Neuropathologic picture with huge numbers of PRP amyloid plaques So as I've said the neuropathology is indistinguishable whether the prions start from cows or humans and the incubation times are very similar The surprise was that these mice are more susceptible to sheep prions than they are to bovine prions natural scrapie from suffix sheep dying in the United States causes disease in about 210 days and the same is true on second passage So we began to think about this in a new way and this is largely Mike Scott's work and Mike began to believe that Perhaps and this is hypothesis still that the BSE strain shown by the little red cube is Present in sheep, but there's not much of it and that its multiplication is restricted in sheep by the faster-growing scrapie strains which are shown by the blue cubes, but that What happened was that during the rendering process The scrapie strains were destroyed because they were more heat labile and the BSE strain survived And now the BSE strain again through the meat and bone meal was passage into the cows on multiple cycles of Passaging as I showed initially due to industrial cannibalism and then eventually high titers of the BSE strain Emerged in the cows and these were path these were and are pathogenic for humans Now let's talk about therapeutic approaches in the last part of the lecture We've undertaken many different approaches and others around the world have taken even additional approaches in a study with Fred Cohn we've undertaken to capitalize on dominant negative inhibition of Pre-on formation that was just covered by Kiyotoshi Kaneko We've used this also in gene therapy studies that I won't have time to talk about And in both of these studies Véronique Perrier who's returned to France and Andrew Wallace who is now in Boston played a major role We've also done some work trying to enhance the clearance of prions and this work was done by Sir Chai Sepadopon who is now professor at Dartmouth. I'll tell you more about the Replication or the inhibition of prion replication by site-specific antibodies and I'll mention a small amount of work About quinocrine and a clinical trial that we've started Now Dennis Burton and Anthony Williamson along with the help of David Parrott's and others in San Francisco But Anthony Williamson and Dennis Burton or it's the Scripps Institute in La Jolla created a library of anti PRP recombinant fabs These bind to epitopes all along the PRP molecule as color-coded here the D13 binds Toward the end terminus more to the middle of the molecule is D18 and R1 and R2 Binded the very C terminus. They're shown in green When David Parrott's added these antibodies at the increasing concentrations to cultured cells producing PRP scrappy What he found was that D18 and D13 were the best D18 being the best of the three and then R1 and R2 Were less good than D13 or D18 and R72 did not work at all We found an excellent correlation With D18 which bound the best and had the highest of it a D with D13 also reasonably good When we did facts analysis looking at the binding of these recombinant fabs to the cell surface And that's shown here the highest curve being the red balls Now what really surprised us was when we did a series of clearance studies and with these clearance studies It eventually became clear to us that We could remove the PRP scrappy with these antibodies and This is simply shown if you look at the gels the panel a for D18 We have one day two days three days and four days and after about a day We see very little signal of the PRP scrappy and This is shown by densinometric tracing in panel B with the red balls That's D18 antibody binding to The surface of the cells and eventually the PRP scrappy disappears Now we know that it Disappears completely because we've done a study where we actually looked at the curing of the cells So in the top panel with they've been Cells have been exposed about for the antibody for a week in two weeks and then three weeks And if we simply look at the D18 in the middle if We now assay at time zero after exposure for a week two weeks or three weeks We see with D18 there is no PRP scrappy if on the other hand we wait a week or we wait two weeks and We've only exposed the cells to the D18 antibody for one week Then we see the reappearance of PRP scrappy But if they've seen the antibody for two weeks or three weeks, it doesn't come back now These experiments have profoundly changed our thinking about PRP scrappy. We thought that PRP scrappy was made of granite Some form of stone and that it never was cleared But what these studies have shown to us is that it is clearly cleared and we now were able to complete a table that was started by David Borschelt and Other contributors to these numbers were Byron Coey and Bruce cheese bro working at the Rocky Mountain Laboratory This was rack around 1990 when it became clear that PRPC was synthesized very rapidly The formation of PRP scrappy was relatively slow three to ten hours PRPC could be degraded with a turnover time of about six hours But it's the degradation of PRP scrappy had to wait until this past summer when we published the paper showing that the Removal of PRP scrappy occurs with a halftime of about 30 hours Now let us turn to the tricyclic drugs and this is the work of Karsten Korth With some help from Barney May What Karsten Korth did was to set out to look for some Nervous system active drugs that might inhibit in cell culture PRP scrappy formation And he looked at many tricyclics that are shown here and the best one was chlorpromosine And if you look at the western blots which are shown just to the left of the structure of chlorpromosine You see that if the presence of five micro molar there are no PRP scrappy bands When Karsten Korth went back and looked at the history of the phenothiazines It became clear to us that we ought to try some other drugs that were seemingly not so related We were non-psychotropic drugs, but were derived from a common precursor the common precursor of both the antimalarials like quinocrine and Chlorpromosine the antipsychotic drug is methylene blue first used by Paul Erlich as a weak antimalarial as early as 1891 in the 1930s quinocrine was synthesized because there was not enough quinine available that was extracted from Chakona the park of the Chakona tree And it wasn't until much later that quinine could be synthesized in the laboratory and Then made widely available. So eventually quinocrine was not used as an antimalarial Now when we looked at quinocrine we saw that quinocrine was five times better Excuse me ten times better if you look at the lower panel on the left of the slide And then you see the western blot you see now we've reduced the concentrations. We have one Micromolar point five micro molar and point one micro molar and at point five micro molar there is no signal And that's is after that's the presence of quinocrine added to the cell culture We made a number of other compounds and these are Barney-Mae's compounds that are numbered BM 51 49 48 47 none of these compounds are as good as the parent quinocrine When we added quinocrine to the cells and then did a curing study We found that quinocrine could also cure these cells and that the PRP scrappy did not return That's shown in the right-hand panel and when we looked at the concentration of quinocrine in some detail It became very clear that at about point three micro molar We were moved half of the PRP scrappy So it was ten again in this study clearly tenfold better than chloropromosine and this number is the same as it from a study published by Dohura in Japan in collaboration with Byron Koeh at the Rocky Mountain Laboratory about a year before we independently discovered this Because we were unaware of their work with quinocrine, but as I said it had been published it was our our Poor command of the literature that we had missed this report Now having found that quinocrine works extremely well in cell culture We set out to study this in mice and we decided that because of the 70-year history of quinocrine And it's treatment of with treatment of parasitic diseases all the way from malaria to giardiasis and the well-documented toxicities in humans of many different ages that we could skip phases one and two of a typical clinical trial and Would easily get approval from the FDA to treat patients with Quartz-Feldeachre disease and that was the case so last summer we applied for approval from the FDA and they gave it to us and also the institutional of course Committees that monitor research and We began treating people now the two that we've had the longest Interactions with our 20-year-old female with new variants CJD who had a modest but we believe significant improvement in her mental status Accompanied by slightly diminished ataxi in Korea, but on Subsequent exam meaning after about 60 days on quinocrine. She began to worsen slightly so We have mixed feelings at this point. These are very hard studies to do of course Since at the moment we have no good biological marker in the blood that can tell us what's happening in the brain When we've looked at her MRI scan, we thought that it was in fact better on quinocrine after two months of treatment in Several cases of advanced CJD where quinocrine has been used by us and by others The patients have gone on to die after the drugs were discontinued because there was really no measurable improvement So what we're trying to do now is to get patients very early in the course of their clinical disease I can tell you that trials of quinocrine are progressing worldwide and I would think within a year We'll have a large body of data So let me summarize what I've said today I've told you about the sporadic and infectious forms of the disease why the wild type protein is converted into wild type PRP scrapy I've talked to you about inherited forms of the disease very briefly where mutant PRPC has converted into mutant PRP scrapy in The inherited forms of the disease the mechanism is clearly a germline mutation in the infectious forms of the disease It is transmission from one host to another and in the sporadic form of the disease, which is the most common We still don't know the mechanism We believe that it is likely to be the spontaneous conversion of PRPC into PRP scrapy or Asomatic mutation Now it may well be that there are very small amounts of PRP scrapy being made all the time the clearance studies that I showed You suggest that that's very possible and that PRP scrapy may have some Physiologic function when it's there at very low levels that we have never appreciated and Then in the disease process this the formation of PRP scrapy outstrips the ability to clear PRP scrapy and under physiologic circumstances and the molecule accumulates and CNS dysfunction follows If on the other hand we use an antibody or a drug such as a polyamine to enhance clearance now the clearance mechanism gets ahead of the formation and When we start we stop the formation and we allow the clearance mechanism to take over and Under these circumstances the PRP scrapy is cleared. I Think that we can take away four truly unprecedented concepts from the discovery of prions First that prions are infectious proteins. They are devoid of DNA and RNA Second that the prion diseases may be manifest as sporadic genetic or infectious illnesses Third that the cellular prion PRPC undergoes a profound Confirmational change when it is converted into PRP scrapy which then causes the disease and even more Heretical is that PRP scrapy may adopt a variety of confirmations each of which specifies a specific Specific disease phenotype and represents a distinct prion strain What I'd like to do is just mention how And how much of an incredible journey this has been for me and For the young people to give them sort of an overview of how it's been to be involved in something so unique and so different Hillary Koprowski gave me this wonderful slide about the four stages of adopting a new idea. This is really a Concept that has permeated biology and all of science for many years and Usually people think about this as the first stage of a new idea and that the reaction of almost everyone is it's impossible The second is maybe it's possible, but it's weak and uninteresting The third is it is true and I told you so and of course the fourth is I thought of it first Now this has been stated in a slightly different way by Lou Thomas Writing in lives of the cell many years ago He said somehow the atmosphere has to be set so that a Disquieting sense of being wrong is the normal attitude of the investigators It has to be taken for granted that the only way in is by writing the unencumbered human imagination With the special rigor required for recognizing that something can be highly improbable Maybe almost impossible and at the same time true Locally a good way to tell how the work is going is to listen in the corridors If you hear the word impossible spoken as an expletive Followed by laughter you will know that someone's orderly research plan is coming nicely along Now one can be more brief and Winston Churchill was able to say it in only a few words Men has occasionally stumbled across the truth, but most pick themselves up and hurry off as if nothing had happened and with that Can you hear me dr. Kruzner? Yeah Okay, are we on Say yes The microphone wasn't plugged in We now have voice do we have voice now? Can you hear me now? Yes, okay Can we hear san francisco? Yes, I I'm here in san francisco. Oh, that's wonderful We have a question here from the audience All our garbage ends up in the ocean or other water sources Do fish have prion diseases? Should we be concerned about this possible source? Well people have people have looked for a prion protein gene in fish And we haven't found one But i'm sure that in sea mammals there are as a prion protein gene and that means there's prpc And certainly there are going to be prion diseases in whales and sea lions And other sea mammals We have another question from the audience. What is the physiologic role of prpc? We don't know the physiologic role of prpc There's a lot of evidence that That has been accumulating To suggest that prpc is a copper binding protein But what it does with copper is unclear Do we have any questions from the panel here while we're meeting for some more questions to come up? Stanley any progress on protein x? Uh The progress is minimal on protein x. I mean what's what's been happening is the data keeps accumulating that protein x has to exist but I don't have any good data for candidates We thought we we thought protein x might be n cam And we carried out a set of studies in collaboration with jerry Edelman and kathryn crossin This is the work of gerald ohm schmitt and gerald's work made it very likely that N cam was protein x but when we took knockout mice for n cam We found that the incubation time was unchanged. So That's not protein x. That's the latest Okay, dr. Fisher Um, how sensitive are the tests to detect whether or not you have prions?   Right. Well, so the immuno assays are not that are currently being used in europe are not very sensitive This has been a constant criticism of these commercially available assays We've developed a new assay and we're hoping to by the By the middle of next year have this assay available for cattle in europe and in fact around the world We have a question from dr. Erling norby Stan howling speaking Congratulations to a very nice presentation in particular the advance of clearing prp sark from tissue cultures by antibodies and drugs My question is you showed a Slide or a picture of the frequency of Med-card disease in the cattle in the uk and the upcoming number of variant cd in humans Yes, do you dare to speculate about where this? Epidemic is developing in man well, I think that most people who look at this are of the belief that The number of cases of new variants cjd is going to keep increasing year by year at least for the next 10 years But it's speculation We just don't know It's clear that the number of cases in the year 2001 will exceed the number of cases In the year 2000, but just how many there'll be by december 31st is not known yet I realize there's a very unsatisfactory answer, but I don't really have a better way to Sort of look at these uncertainties my my guess is early that in another year or two The slope of that curve will become clear Thank you Do we have any other questions from the panel here? If not, we have another question from the floor here Have any chaperones been targeted as points of control in the transport of prp sc? So the answer is That at the moment there are no good candidates What is clear is from the study of yeast prions That the work of sue Liebman and susan lin quest and urie churnoff and others have shown that For the psi prion That a chaperone is involved in the formation of the psi prion So there's clear precedent for a chaperone Being involved and of course that would be the protein x that we're looking for a protein that Helps convert prpc into prp scrappy Is there another protein then to which prp scrappy binds that facilitates its transport and thus its infection of other cells That's a very good question And we have no knowledge of that Either in yeast or in mammals Another question from the audience Is the mechanism by which uh, qunask Is it qunasa churn acts known Is if so, are there any known effects of it on prpc? Yeah, we we don't know Anything about the mechanism of qunokrin action. We know that prpc expression is not suppressed by qunokrin So there's prpc being made We do not see qunokrin binding to either prp scrappy or prpc And the so the answer is we don't know the mechanism of action to qunokrin And we're trying to begin to decipher this because I think that would be useful There are obviously some other proteins involved that we don't know about That qunokrin or a metabolite of qunokrin binds to Is another question from the audience Is there a hypothesis why young humans are susceptible to new variants cjd and not older humans? Well, I think that the hypothesis is that For reasons that we don't understand at all The younger people Are able to transport the prions and multiply them in their lymphoid system more readily than our older people But why that is We have no idea at this point Okay And another one how do prions induce the misfolding of wild type proteins? So we don't know much about the mechanism by which prpc changes into prp scrappy or the mechanism by which The sub 35 protein is converted into into the cy form in yeast In yeast though the mechanism is somewhat different than it is in mammals and yeast this occurs in the cytoplasm of the yeast cell and Many features of the yeast conversion process can be modeled With either peptides or the whole protein in a test tube That's not true in the case of the mammalian prions Although there are some people who claim to have been able to create the modification create the multiplication of prions There has not been good confirmation of that work In The in the mammal as I showed you This process occurs in a very specific cellular compartment It's the raft the lip the cholesterol rich microdomains or when the rafts coalesced to the form these cavioli So they're probably they're probably One or two or three proteins i.e. protein x that are involved in this very special micro domain In the conversion process, but the mechanics of this we don't understand Yes, uh, so John Maddox would like to ask you a question Stanley, um, you we first met in 1981. You may remember that you see us often and you kindly talked to the nature conference some years afterwards But I wanted to ask was about the situation in britain where a hundred people so far have been infected with cgd As I understand it, there's a polymorphism in the protein at amino acid 27 in which valine or methionine substituted for each other At present all the people infected in britain have been The more susceptible hemozigate, which I think is methionine For one thing this is the question uh, for one thing How did it come about that? The different proteins Behaved differently in these circumstances. Is it that the substitution Enhances the folding weight one or the other and a second On the question of What what actually happens? What on the question of the mechanism? What's being done to find out how it is that one protein can Uh, a misfolded protein can in fact induced a misfolding of others Well, let me try to let me try to answer the polymorphism question first The polymorphism is at Residue 129 and you're absolutely correct. It is a methionine or valine Uh, just to put this in a little perspective About 35 percent of the population are methionine methionine About five percent are valine valine And the rest of them are methionine valine The it's not surprising that A very large group of people That one can look at are Turn out to Have all methionine be methionine methionine in these hundred cases in the sense that That about a third to almost a half of the population are methionine methionine But now why is that? It it looks as though and a lot of this work comes from john collage many years ago that most people with a sporadic cjd are methionine methionine or valine valine And that if you are heterozygous you seem to be protected And that's probably because A protein with methionine a prp scrapie doesn't interact well with a prpc that's valine And vice versa So it's thought that if people are going to come down who are methionine valine With new variants cjd that it will be much later Now coming back to methionine methionine. Why aren't they good? Why don't we see some valine valine people? Well, it may well be that the strain of prions, which is very unusual as I showed you in my talk where The strain of new variants cjd. This is a human prion Transmits into bovanized mice much better than humanized mice in contrast to all other human prion diseases that we've looked at That this particular fold Does much better in replicating itself on a methionine backbone meaning a 129 polymorphism with methionine Then it does with a valine And the opposite probably is true with growth hormone. So iatrogenic cjd induced by growth hormone They're a very large number of valine valine cases So I my guess is that this is a strain issue now all of this is bound up in your second question of what is being done to decipher this mechanism I think people are looking for auxiliary proteins People are trying to develop an in vitro conversion system that actually produces large amounts of infectivity But that has not been successful because such a system would allow us to dissect all of the components People are trying to get information about the structure of prp scrapy And until the two-dimensional crystallography or what is called electron crystallography that I showed you today Where we're using this to try to constrain the models and to get much better EM images for image reconstruction By using a much better microscope now that we've just obtained The only data that I know of that was available about prp scrapy structure was that derived from antibody studies using the panel of antibodies that I showed you along with others and also the optical spectroscopy data using cd and Fourier transform infrared spectroscopy, but I am unaware of anything else that is being done at this moment There are attempts here in the Bay Area, Alex Pines and David Wemmer to use solid state NMR on peptides meaning 50 MERS that induce a genetic form of prion disease in mice But we really lack the technology to Get at the structure of an insoluble protein Uh, which is not made in enormous quantities in in a mammal. This is very very difficult Okay, another question from the audience. Excuse me Is there any similarity in brain pathology in any of the three varieties of prion diseases and that found in alzheimer's disease? So the answer the answer to that question is there are extreme similarities Uh There are forms of prion disease one of them studied by martin farlow and bernadino getty in great detail at the university of indiana This is an india what is called the indiana kindred that have amyloid plaques That are surrounded by neurofibrillary tangles now these amyloid plaques contain prp amyloid and not the a beta of alzheimer's disease So here the pathology without antibodies is truly indistinguishable from alzheimer's disease Okay, just a couple more questions here from the audience Is the appearance of bse in the united states inevitable? I think so Uh My guess we don't in the current japanese case, which is probably the most instructive to talk about We don't really know the origin of Bse in the cow that was diagnosed as positive Is this a sporadic case of bse? That occurred like sporadic cjd or is this case the result of feeding practices? Where there was some contaminate prion tainted meat and bone meal or some other form of feed that was brought from europe and then Transmitted the prions to the cow the answer is we don't know but my guess is that in the united states as We begin to look more and more as the tests get better and better for prions That we will find cattle that are infected now Are they infected from europe the answer i think will be no And in the case of the japanese cow the answer is we still don't know Another related question in the u.s. There's a disease similar to scrapie that's been described in elk and deer Does this involve a similar mechanism and could the hunters be in danger of being infected? All right, so the disease that's being talked about is chronic wasting disease It's found in mule deer and elk and whitetail deer throughout the rocky mountains in the united states and then in The rocky mountain range in canada This is a prion disease. They have large numbers of prp amyloid plaques in the brains of these animals And does this disease pose a threat for humans? The answer is we don't know there have been in recent years Uh three or four young people who were deer hunters who came down with kreuzfeld jacob disease And the answer and we just don't know whether there's a relationship between Their cjd and that and the deer in all of these cases we suffer from the same Lack of expert technology. We have very we have a very difficult time saying that a prion in any individual Species came from another species or came from another source And that's because there is no nucleic acid. So there's no unique genetically Determinable material That is such as a virus Where we would find the virus in the mule deer or in the elk or the whitetail deer and now find it in the human instead what we have is a Protein that we all have which has now changed its confirmation in the mule deer or the elk or the whitetail deer We have a hunter with cjd and was And and was the person that person cjd due to The ingestion of prions from the mule deer or elk that now stimulate the conversion of human prpc into human prp Scrapey we can only begin to decipher that through understanding the conformations of these proteins And as and as I answered to john maddox In the previous question the technology for all of this is so primitive Even though it's so elegant when you have a water soluble protein, but so primitive with these insoluble prions that We have many many Years of research to do and new technologies are needed to be able to answer the kinds of questions that have been posed Okay, do we have any other closing questions from panelists here? Then I have one last question from the audience Do you eat beef? The answer is yes, I eat beef, but it's geographically restricted Very good. Thank you so much dr. Prugner You


Rwy'n arvanedd nefnol. D 밤 independent. Pawn eich accuseysau Ratford, 고� depends, a gallwn ymwneudu I Unlessedd y Llywodraeth. YmprGaith Cymru.'s Itim 3 i hwn sekirbeid 영상. Bu音 T1AU Alison Harris. Agenda item 2 is consideration of an instrument subject in negative procedure. We have council tax reduction, Scotland amendment number 3, regulations 2018. These regulations make further amendments to the council tax reduction regulations 2012 and the council tax reduction state pension credit, Scotland regulations 2012, which are the principle regulations. There are two issues for the committee to address. The first issue is a drafting error relating to the definition of, quotes, an error on a point of law contained within regulation 4A of the instrument. The error has been acknowledged by the Scottish Government. Does the committee wish to draw the regulations to the attention of the Parliament on reporting ground I as there appears to be defective drafting within regulation 4A? Secondly, does the committee wish to recommend that if the Scottish Government assesses that this drafting error could have some unintended effects, it may be preferable to bring forward an amendment to correct the error promptly, given that the provisions will come into force on 28 November. The second issue relates to the question of whether the regulations raise a devolution issue, as they may relate to matters that are reserved by section F1 of part 2 of schedule 5 to the Scotland Act 1998, which concerns social security schemes. As the committee knows, bios has been considered each time the council tax reduction regulations are amended, and this is the 15th amendment of the principle regulations. For the five amendments considered this session, the committee has agreed on division to report that the regulations raise a devolution issue. The committee recognises that the Scottish Government takes a contrary view. Since September 2016, a new exception 10 to the social security reservation has given the Parliament powers to create new benefit schemes in areas of devolve responsibility, where the requirements of the exception are satisfied, including that the new scheme must be funded from the Scottish Consolidated Fund. The committee suggested to the Scottish Government, when reporting on SSI 2017-41, that framing a new scheme in consolidating regulations could remove the committee's concern, were that scheme to comply with the requirements of exception 10. The Scottish Government has indicated today that it has no plans to undertake a consolidation. It seems therefore unlikely that the difference of view on the devolution issue will be resolved in the immediate future. As the committee's position on the devolution issue and its possible resolution is clearly stated in its previous reports, does the committee consider that it is not necessary to repeat that view again and for this to be explained in the report on this instrument? Does the committee wish to continue to encourage the Scottish Government to undertake a consolidation of the principle regulations within a reasonable timescale in the interests of clarity and accessibility? I will move the meeting into private.


Hello in this lecture we will define investing activities. According to fundamental accounting principles Wild 22nd edition the definition of investing activities is transactions that involve purchasing and selling long term assets includes making and collecting notes receivable and investments and other than cash equivalents. Support accounting instruction by clicking the link below giving you a free month membership to all of the content on our website, broken out by category further broken out by course each course then organized in a logical reasonable fashion making it much more easy to find what you need than can be done on a YouTube page. We also include added resources such as Excel practice problems PDF files and more like QuickBooks backup files when applicable. So once again click the link below for a free month membership to our website and all the content on it. When considering investing activities we are looking at them in terms of the statement of cash flows this being a worksheet related to a statement of cash flows form having three parts those three parts of the statement of cash flows including cash flows from operating activities cash flows from investing activities and cash flows from financing activities we are focusing here on cash flows from investing activities note that there are two methods we could use for the statement of cash flows a direct method and an indirect method when we're considering those two methods we're usually concentrating on the cash flows from operating activity generally the largest part of the statement of cash flows basically converting an income statement to that is on a cruel basis to the income statement which will be more on a cash basis and under the direct method we would do that directly basically converting line items and under the indirect method we would do that by starting with net income looking for those differences to arrive from net income to net cash flows from operating activities it's important to note that the other two sections cash flows from investing activities cash flows from financing activities will be the same under either method whether we be using the direct method or the indirect method these being items that will have cash related to them cash inflows and outflows that are not part of the normal operations cash flows from investing activities would include those things that we would assume in investing activities including things like us investing in other company stocks and bonds as well as receivables that we would have from true investments traditional investments we might think of from an individual standpoint in terms of when we invest the thing that's also in investing activities which can be a bit tricky in terms of terminology will be investments in property plants and equipment and the reason that is is because when we put money into property plant and equipment say we buy a machine or something machinery that is an investment in a form of investment the term investment can be a bit confusing a bit tricky because we can use the term investment to mean different things in different contexts when we talk about financing off time or for our personal finances we're usually thinking of the term investment in terms of of stocks and bonds types of investments but note that any long-term asset anything that's going to help us generate revenue in the future in terms of a business perspective in terms of the goal of the business to generate revenue is a form of investment so when we're purchasing long-term assets property plants and equipment those are a type of investment therefore those will go into the investing activities we know that they're not going to be in the operating activities and just so you can memorize this and see why they're not up here note that the normal journal entry when we purchase equipment is not going to involve an income statement account it's it's going to be on the books as an asset and we're going to pay cash or take out a loan we don't have any revenue or expenses therefore it's not going to be in operating and it has to be somewhere in investing or financing and we're going to put it in investing because it is an investment into the future even though it's recorded on the balance sheet under the asset section which could be thought of as a type of investment pretty much all assets are a type of investments that's why we have them in order to help us generate revenue in the future