


S1: okay a few announcements... a few announcements. before we begin i get a lot of email questions and you can keep telling me lots of questions about, project number three. just to remind you, for project number three which is due on Wednesday... <WRITING ON BOARD THROUGHOUT NEXT 1:04 OF UTTERANCE> you have, to basically combine everything you learned from project one and project two. ultimately that's the goal. the idea is that instead of looking for a, homolog that is only homologous, with amino acid residues at particular places in a protein, you are now looking at, homologs in space. so it's spatial or structural homologs that are the focus for project number three <P :04> you use the DALI server. the DALI server superimposes two, three-dimensional maps right on top of each other. the problem with the DALI, is unless you have the proper plug-ins on your computer that you're using, you can't print directly from the DALI server. so you download the file, that is, the three-dimensional superimposition you download it, you give it a dot, P-D-B, and what_ i think most things that you download are given for whatever reason, the name Q-Z, i have no idea why don't ask me why. but the P-D-B has to be appended to the end of the name so that RASMOL will recognize it. download it directly into your folder that carries RASMOL on your computer, and then you have a kind of abbreviated three-dimensional layout. so it's really important that you recognize that the superimposition is not, the combination the straight linear combination of two of these files, because in order to superimpose things in the space you have to move the positions, of all of the atoms of the U-one protein, to meet up with the atoms of the other protein. so these are not either of them this three-D superimposition, they are not directly, the same, RASMOL files for the indivi- individual proteins okay? is that clear? you cannot often, manipulate, this thing that you call a downloaded three-D superimposition. alright? you can't manipulate it. you can only print this in <WRITING ON BOARD THROUGHOUT NEXT :06 OF UTTERANCE> backbone, because it is not a continuous file. so, unless you're very lucky, and you just happen to pick proteins that can be very much, uh conserved when they're put into the three-D superimposition, you will have a discontinuous point-by-point array that shows the three-D overlap, and you only can print that using backbone, or i think points. i'm trying to remember what it is. you can do, individual points that don't help you at all to trace the backbone of the molecule. so print that in backbone. you are only required to print, one, superimposition. if you wish you can print more. you can select things in that overlapped view if you want. but often it's harder to deal with this particular file, rather than going_ once you download the three-D superimposition, be careful to write down, what the P, P-D-B file number is for the other protein. you have to check it off in order to get it superimposed. so check that other, <WRITING ON BOARD THROUGHOUT NEXT 2:30 OF UTTERANCE> P-D-B file number, okay? that's important. you have to use that as an extension code, then you go to the P-D-B searchlite. you plug in that, P-D-B accession number then you use RASMOL on this, three times... to highlight residues that are in your paper. because the other thing that the P-D-B searchlite gives you when you plug in your accession number is a primary citation that tells you the paper, that shows you where the paper is what article it was what journal it's been published in, that dir- that is directly, the report of this guy's structure. whatever that protein is if it's that structural, paper. then you can get the citation there, if you're very lucky and some of you i think have gone to great pains and at great lengths, to make sure that you only get a paper, that you can download directly. that's okay, just anything to avoid going to the library isn't that true? is that what you guys are doing? anything_ no okay. some of you are not (out of the loop.) no, not too many. i have a feeling i'm gonna see a lot of Medline reports_ structural paper, you should have, you do not need to turn in the paper, but make sure that you give me the_ you can a- actually just printout, this searchlite um, that's as good as a (FAFSA) report actually. if you just print out that page that has the citation in it. one page discussion, and that discussion should include, the three pictures... i wanted you, highlight the certain (things) <NOISE DISRUPTION> in those pictures. some good examples would be highlight, the catalytic, residues. what does your protein do? some of you will have a protein that's a homolog but only overlaps, with a very small portion, of your protein. o- of S-Q-D-one. they only overlap a small portion, that's fine. some of you, pretty much the whole proteins will overlap. that's fine too. you don't have to necessarily highlight what is conserved, spatially, if everything in the protein is conserved except that little loop, in the, S-Q-D-one right? i mean just highlight the whole, homolog so, keep that in mind. catalytic function, whatever... structural analyses, you hafta read the paper, to do that part... any questions whatsoever on this? 
S2: is it okay to have one area of sequence homology or do you not want that at all? 
S1: oh that's fine. absolutely you mean highlight it? 
S2: amino acid homo- homology. 
S1: yes but did you me- are you saying that you have one sequence highlighted on your, protein? that is an amino acid, sequence homo- homo- ho- <SOUND EFFECT> homolog?
S2: yeah. even though it's structurally it's um amino acid also?
S1: it's not necessarily. it would depend did you do multiple line?
S2: yeah.
S1: okay. so, it depends if you have done multiline, you wou- might be dramatically disc- uh suh- actually dismayed, to find that a lot of your proteins are not, sequentially, homologous. 
S2: but the G-G, X G 
S1: yes that's fine. and if you wanna highlight that that's great. you are not required i don't believe i dropped multiline out of the requirement, because it would take a lot more time, to do a sequence, analysis of your proteins. but if you want to, don't be amazed, if your proteins are really not, homologous in terms of their sequence. it's the space, that, that's the_ that's what the DALI server is doing is showing how they look in space. any questions whatsoever? i haven't even been on email yet today just in case you're, wondering why i haven't replied. i have this, haunting suspicion that there's lots of email so i hope, i'm getting a lot of your a- of your questions answered here, which should do that. yes Andy? 
S3: by looking at the two overlapping structures, how are you supposed to determine where it is structurally homologous.
S1: by looking at the two overlapping where you're supposed to t- 
S3: when you have the superimposition picture, would it how does it help you determine where each molecule (itself) (xx)? 
S1: you're gonna hafta guess, where they're homologous. that's what i'm saying, if you've only got a small section you can probably trace the backbone. if you've got the whole overview look for things in S-Q-D-one that you're used to, um identifying. look for the (Rawson) fold. if you can see where that is then you can, see where yours is with relation to that. you can in many cases select things that you've already selected in S-Q-D-one. you can select them in the overlap, view. and that might also give you some idea as to what is actually homologous. if you know for instance the se- the sequence, of amino acids that make the (Rawson) fold, select it, on your_ really overlapping, see whether that's where the homolog, is positioned. but taking a guess is just fine i mean the overlap will show me, where the homology is. so don't worry that you hafta get exactly that residue, on any of your RASMOL. i don't care if you don't highlight that at all. and highlight other specific things that you think plays an important part of function or structure, of your homolog. okay? okay remember there's an exam on, yes? Friday 
S4: did you want the article with the, with the report? [S1: did i want the ] do you want us to put the article (with) (xx) 
S1: no it's just gonna make_ you know don't kill any more trees [S4: <LAUGH> okay ] to make me an extra copy of that. think of all the plants and what they do for us, with this (bill) and everything. exam Friday ten to twelve your last exam. that's cause for celebration alone isn't it? and you might think that's why the bagels are in here today, but they're not. they're in here because this is the lecture on, insulin and glucagon, so i am feeding you to help you see, firsthand, the effects, of hormonal signals. okay? that's the key. so, again, let's make this real casual you come down here anytime and, help yourself to this, this wonderful feast. there's a lot of bagels left so, feel free. to come down at any time. let's review. this by the way is a great diagram it's on page seven-fifty-six, in Leninger. let's review what we've looked at so far. for all of our studies to date, the catabolic and the anabolic, pathways, we've pretty much been focused, on a few different kinds of cells, in us. those kinds of cells are the liver cells or the <WRITING ON BOARD THROUGHOUT NEXT 6:16 OF UTTERANCE> hepatocytes which has a very extensive role in all catabolic and anabolic function we focused on muscle cells which, also have a very dramatic role, in anabolic and catabolic regulation, and to some extent we've looked at, fat cells. storage, diglycerides, and let's just review again what's going on here. in the liver cells, what's the liver do? what's the major important thing that the liver does? it's the major place for production, and distribution, of what? 
SS: glucose 
S1: glucose, that's one thing. glucose production, main goal. so it does a lot of, glycogen breakdown it does a lot of glycogen synthesis, which is (xx) a major storage place of sugars. it does a lot of glycolysis well actually relative to other cells it doesn't do a lot of glycolysis. it does far more gluconeogenesis. it doesn't necessarily need a lot, of metabolic, you know input, to keep its own system running. so its major focus is the production of glucose. and the production of glucose, because it is a central molecule can happen, both from glycogen breakdown, and from... gluconeogenesis. that is the reproduction, of, glucose from, wherever we've stopped the pathway. pyruvate, even beyond in some organisms. and that's called <WRITING> gluconeogenesis, </WRITING> and what's a, premier example of this? lactic acid that builds up in these cells, can't go right back into, glycolysis, whatever (lean) muscle cell. they have to be shipped, lactic acid molecules have to be shipped all the way to the liver, liver can turn it back into pyruvate, and who wouldn't take pyruvate through those three bypasses, <SNEEZE SU-M> that's_ god bless you, that's what's really noticeable, and notable, of the glyc- uh gluconeogenis- gluconeogenic pathway. it's the fact that it's pretty much the same as glycolysis, (that) there's only three, different enzymatic reactions in glycolysis, having to be reversed with the use of bypasses... can you name me the bypasses? do you remember the first bypass? it's that really ornate one that goes out into the mitochondria, out of the mitochondria, that's a very complex one, with the pyruvate carboxylase, and two other enzymes the malate dehydrogenase. so that's the first very ornate, bypass. the second bypass would be, which one? it's the bypass of P-F-K-one. so it's done by F-B-s- F-one-six-bisphosphatase, more than a mouthful. you guys i know, just remember the, abbreviations are (define) F-B-P-ase. what's the third and final bypass? [SS: hexokinase ] hexokinase hexokinase, bypass is what? <P :05> what is the enzyme? hexokinase doesn't produce itself... what's it called? glucose-six-oxidase. kay? that's the bypass. alright and, very notable, the liver's the only, set of cells, that carries the final bypass. this is, probably the most important one in distinguishing liver atrophy and muscle atrophy. okay? huge glycogen stores, what else is the liver involved in? what else is the liver involved in? it's the garbage, collector. it's involved in sending out, sterols and in picking up sterols and fatty acids, you know H-D-L (and) L-E-L you should know that whole trafficking idea, let's just call it sterol (glycogen) trafficking. so it's got a very big goal. and that's just the liver. muscle, is dramatically different. what is muscle's main goal? activity, that's it's main goal. all activity all the time even when resting it's actually active by most cellular standards. so it has, very little glycogen stores because think of the mass, of the muscle cell. you know, they're not big in size they're not a big organ, individual set of muscles for instance. so it has very little glycogen stores... it breaks it down, in times of activity. immediately to glucose-one-phosphate and glucose-six-phosphate. it is brought, and the skeletal muscle for instance goes directly into, glycolysis. so the goals are very different at this point. the main goal of the liver is to produce glucose. that is not so, of skeletal muscle. more activity, more breakdown, it does very little gluconeogenesis by itself. in fact what would be its point to, making glucose? what would be the point for the muscle to make glucose? 
S5: to use it? 
S1: to use it, it's already using it, there would be no point to reverse that. so it's always (gonna do) this as much as it can. but it doesn't do the muscle any, benefit, to be able to reverse all of, glycolysis. yes? 
S6: would the muscle create glucose if the blood gl- glucose dropped, precipitously and there was like danger to the whole organism or something? 
S1: no, be- w- it might, it might reverse this to some extent, but it would never be able to send it out to other cells. [S6: oh that's right cuz it doesn't have the hexokina- (xx) ] it has no, bypass. it has no bypass so you would never even if it can reverse, with the first bypass, the pyruvate bypass, it could never send it all the way, to glucose itself. whatever glucose is in there is trapped in there, and that's why muscles have another storage molecule, <WRITING ON BOARD THROUGHT NEXT 1:56 OF UTTERANCE> which i am sure any of you who have, ever been to the, General Nutrition Center, have you have you seen it? the General_ creatine, or creatine phosphate. it actually is a very, similar molecule to one that we've looked at, before, uh, (xx) it doesn't really matter (xx) i think this is it what this molecule allows the muscles to do is (xx) <NOISE DISRUPTION> allow glycogen storage, it allows itself to become phosphorylated directly, using A-T-P (xx) creatine kinase that's (creatine-M-I-A-six-phosphatase) on the terminal nitrogen so what this does, is just continue the balance, of this enzyme's activity, it can store, a lot of potential energy in creatine phosphate. because if the enzyme gets a total amount of this that's rising, it can drive its reaction forward, or backwards as long, as the levels of this increase the equilibrium of this reaction will be shifted, and it'll drive this, in this direction and what happens as as a result of driving this reaction? you make A-T-P. this is, one of the major stor- storage molecules, in muscle cells. and they can (slow out) this concentration is very high in muscle cells. and in, just by shifting the equilibrium, harvest A-T-P, enough A-T-P to keep their muscles going, even without having to go through this whole pathway. so lactic acid is a way that muscles have developed to, bypass the need for N-A-D-plus. you remember lactic acid in the Cori cycle? muscles who are working very quickly, can't afford to have to wait all that, you know, aerobic, kind of exercise. they have to be able to regenerate N-A-D-plus very quickly in order to keep just, this part, going. so, lactic acid builds up and you've regenerated N-A-D-plus. <WRITING ON BOARD THROUGHOUT NEXT 10:15 OF UTTERANCE> just a reminder, Cori cycle, that's where the formation (this,) so that glycolysis, can keep going. this, whole idea, this enzyme which is very heavily regulated by hormonal influences, <CORRECTING WRITING ON BOARD> influenseses,(sic) you know what i mean. a hormonal regulation that's what causes this kind of storage to be able to keep the muscles going, even when they're not being fed glucose, for great periods of time <P :04> different roles, completely different roles. same with this one, a different role in general. it's mostly storage of fats fats are not the premier, compound for metabolic purposes. they are something used when, sugars are in short supply. all these cells have different hormonal signals targeting them. because they have different roles. mainly from liver glucagon is the major hormone, that targets the liver. i'm not so concerned that you, read the first part of chapter twenty-two for information that, would be on the exam. that is what cells produce all of these things the pa- pancreatic D-cells those i'm not, concerned with. i don't care that you know exactly which part of the brain or which gland produces any of these things, but you should know which ones target which kinds of cells. glucagon major, major influences are probably the cells. that's where the biggest pool of glucagon-type receptors are. muscle cells, epinephrine adipose cells, adipose tissue, they are, pretty much glucagon, but epinephrine effects can can hit them too. insulin insulin, affects all of these. insulin will have a different kind of affect, on all of these cells. it targets all of them. that means you will find insulin receptors, on all of these types of cells. somatostatin somatostatin is another, hormone, that also, its receptors, that that hormone will be felt in all different kinds of cell types vasopressin... the blood pressure one... that targets these cell types and specifically, the cells that bind the capillaries and things that's when that's why (this) has the most bla- blood pressure effects. it's also involved in the kidney functions, so kidney cells those have a lot of, vasopressin-type receptors. it's the blood pressure (one.) okay, what is somatastatin's effects? somatostatin is what actually regulates glucagon and insulin together. yeah?
S4: is vasopressin a, hormone?
S1: it is, yes... yes, <WRITING ON BOARD THROUGHOUT NEXT 5:04 OF UTTERANCE> (xx) here we go. somatostatin, is mainly involved in the balance keeping the balance on the insulin and glucagon signals. so what kind of ordinase is signaling others when the glucagon especially, in those cell types that would have both signals going on. for instance which ones would have both insulin and glucagon? <POINTS TO BOARD> this one. this one. not so much in the mesa- muscle cells right? because glucagon glucagon isn't going to really target those muscle cells. so this one is basically, regulates... insulin... with and, versus, we can have it as a conta- as a competition. insulin versus, glucagon. and it's mainly, in liver... okay? so let's talk a little bit more, about what, these coordinating, signals do... <CHANGING BLACKBOARD> don't be worried it almost never falls. i've been told that. okay hormonal signals, how does a hormone work? it is produced by very specialized, cells... in organs or in glands. so we're mainly talking about hormones, like, the peptide and the amine hormones. okay we're not talking about, those steroid-derived hormones that are locally produced and locally effectors. they are transported through the bloodstream, which tells you a little bit about their solubility. they target, only those cells that have specific receptors. these are of course, i better put it up here, peptide, and amines, because at the very end we talked about steroid hormones, and those have a different mode, of activity. specific receptors that means they don't just get picked up from the bloodstream wherever there's a low concentration, and have their effects in their cells. they actually target only very specific cells. so when those glands turn on the production, of these, hormones, they're going through your bloodstream (at) high levels. and, turning off that signal means that it will take some time for the bloodstream's, hormonal levels to decrease. there is a turnover time involved. so with every hormonal signal the key has to be, and for every signal at all for every signal in every cell all the time, the key to signalling, is that there has to be an on, and an off switch... otherwise it's not a signal. otherwise it's just a continuum. so the regulation, of all these hormones, is involved with how to turn on the signal, and how to turn that signal off by itself there has to be to some extent the autoregulation. and that's what we focus on. now if it is a signal, it somehow, is a pulse. it is not a continuous thing. so that, what would be the point of signalling then you_ then you'd have to have another signal come round to turn it off. consider autoregulation. that's really important. and how do cells determine, what effects those particular hormones will have on them? well the individual cells that are targeted by hormones, can choose, how to have those effects. and one of the ways, that cells can choose is, by putting what receptors it has on its surface. if a cell produces a receptor to a particular hormone, that means that it wants to be turned on, or off, by that hormone's activities. the cell types can regulate, hormonal influence that's one word, influence by... having receptors... or not having receptors, having receptors at high concentration on the cell surface, or in low concentration where have we seen this before? having nothing to do with hormones? what kinds of cells can say, kind of tailor oh i wanna have this much of that receptor on my surface or i wanna bring it in and hold it within the vesicle so that it can't be targeted...? you're whispering the answer, what? whatta you think? no, you don't know? 
SU-M: the cholesterol for (xx) 
S1: yes, L-D-L receptors. okay, they can con- they can determine, how many receptors what's the concentration. so in these cells, how many receptors are you going to, transcribe, translate and make on the cell surface. they can also change, the kinds of second messengers, that are turned on by these receptors and we're gonna talk about that in a minute. and they can ultimately determine, the kind of effects that these hormones will have, by choosing which enzymes, are going to be turned on ultimately by the second messengers. by selectively having enzymes like glucose-six-phosphatase, or choosing not to have that enzyme, like the skeletal muscle. what enzymes it has, those are the ones that will be affected by the hormonal cascade. the skeletal muscle has creatine kinase. the l- the liver does not. okay? these are the these are the individual enzymes that are, that are encoded in that cell and produced in that cell. so, just as a side issue because i didn't i didn't say enough bad things about it. in the name of equilibrium do you think eating or ingesting large amounts, of these things, can help you? and yet you can buy it at huge and enormous cost. and, it's very popular, among certain classes, of, <SS LAUGH> of individuals, to, <SS LAUGH> to actually to eat this in huge quantities. but, i just say to you equilibrium is the key. you can't force in more than is there. okay? G-N-C at least (xx,) you know other than the basic, vitamins that you can get there, i just, every time i go in there and i see those huge things of amino acids, and <SS LAUGH> i (end up) thinking, who pays, this kind of money, for like something you can just eat more of a certain vegetable, and you'd have the same effect. it all goes out in the name of equilibrium say that, in the name of equilibrium. <SS LAUGH> you don't wanna say that? okay, you're not gonna e- you're not getting light-headed yet are you? from all those, simple sugars that you just digested? no...? i've i've made you all pass out. you can all come up and get another one. <SS LAUGH> you can. yes Ashley? 
S7: i have a question about this whole creatine business [S1: yeah ] that if a bodybuilder is eating the creatine right? what is the [S1: or ] point [S1: the creatine phosphate i think you can get either one. ] okay, so he's eating 
S1: which, i mean it astounds me. if it's creatine phosphate that you're eating, what are the chances that it's gonna be uptaken? that the phosphate (sycon) i mean that's the whole key, to glucose transport. you can't get the glucose phosphoryl- phosphorylate (into cells) so (it's) just 
S7: is it broken down in the stomach because if it's all the acids (and stuff) 
S1: well that's another thing. i mean look at, look at what's (going on in this stuff) does anybody see the tail end or the top end of this? what does it look like to you? [SS: protein ] it looks like it's waiting to be turned into urea. <SS LAUGH> (there you are,) (xx) someone can see it there it is and it's a mess/ you tell me, not only are they gonna have to bypass the digestive process to get into the, right? you have to. there would have to be presumably some kind of, transporter that would recognize it as something that a skeletal muscle could use. you tell me if that's possible, and then you would have to go and and the idea that you can stockpile it by eating it. just like, it goes against every major, natural law of equilibrium that there ever could be. okay? so don't buy that stuff, if you are. [SU-M: why does it work then? ] it doesn't work. <SS LAUGH> it doesn't work. there's no metabolic reason that it would work there is no metabolic reason. does it mean if you take it it doesn't have a kind of a placebo effect? sure. [SU-F: ah ] you know you work out longer, you got all that extra energy, <SS LAUGH> you know that's all that's all that's all it is, it's amazing. <SU-F LAUGH> what the mind can do. yes Darrin? 
S8: how about if you couple it with glucose? does [S1: the what? ] (it work?) if you couple it with gu- y- uh glucose? 
S1: with glucose? whatta you mean? in one ingested form? 
S8: no, if it bonds to glucose then, wouldn't the cell pick up the glucose, (and) then pick up the creatine? 
S1: not, not likely. [S8: oh ] you'd have to have a particular transporter that would be able to see not only the glucose but the, the creatine that's attached. it's not it's not by itself, working in the way that, the label says it will. on the other hand if you do take high quantities of, things like chondroitin and things like this, i don't know how familiar you are with the G-N-C things. you can do some kind of ligament buildup. you can do, some things along those lines. but the metabolic basis for it is not known. it's just known that if you take certain_ it's the same thing with vitamin therapies. you can increase certain levels of vitamins and that can help a lot of the neurological processes a lot of things, that that the me- the metabolic basis isn't known. what the body is doing with it isn't known. but it's been correlated with increased ligament production and increased rebuilding (and) regeneration. that's not to say it's bad it's just not, it's not benefitting you, in the way that you think it is. it can't, a little bit of biochemistry can be anybody's nightmare who goes to, G-N-C or, who goes to a pediatrician, or ever, do have (a pediatrician.) that will all (xx) worst nightmare. and you can do that too. <SS LAUGH> you guys have to worry about those things okay? that was a little sideline. but it was still fun, wasn't it? so let's go on to what is actually going on. are you feeling the effects of what you've eaten? what are you feeling? you tell me what're you feeling, those of you who actually started chewing on these things. shall i send them circulating? should i send them circulating? can you pick out of the bag? i can't circulate the, cream cheese. what are you thinking? are you feeling sleepy? yes, you're feeling sleepy do you know why? do you know why? cuz you are in, debt. because you're thinking to yourself, wait a minute i just ate. and i've got all these carbohydrates and they should be circling and they are circling. except the energy that you expended in chewing, has put you in debt. okay? <SS LAUGH> it has put you in debt. <WRITING ON BOARD THROUGHOUT NEXT 11:55 OF UTTERANCE> you have eaten... and also if you haven't eaten for a while before actually (maybe) you bite into these things, you're thinking to yourself okay (so why) you've eaten basically simple sugars. that's basically what the carbohydrate that's that's the bagels here the cream cheese i mean, sometimes you have to make those simple sugars taste a little bit better. <SS LAUGH> but you have eaten, simple sugars. but you chewed them. and bagels are notoriously, chewy. and the chewing process, has not only probably made your jaws ache, depending on how many you've eaten, but has actually put you in a little bit of skeletal muscle de- debt. and why, if your jaws are aching, why are they aching? because you were chewing so quickly, that lactic acid was building up there, and now it's being transported, to the liver, for regeneration, to the glucose. okay? but, the good thing for you is glucose is on the way. because your, stomach has started, digestive processes. and as a direct result, of eating this, ultimately, within, a half an hour, probably, you will have a much elevated glucose concentration. and that elevation will be seen. and it will be seen immediately. and as the high blood glucose increases, insulin, is produced by the pancreas... it says okay, i've got, a lotta glucose... insulin is sent out. and insulin targets every single cell, that contains an insulin receptor. and insulin receptors are pretty much, the easiest kind of receptive systems. and we'll s- start with those... so where're we? we're at that first level, of regulation. only the cells that have a receptor, will feel the effects of what you've eaten. what does the insulin, receptor look like? (there's this) nice little drawing... on page, seven hundred and seventy in Leninger, and i'll draw it in a cartoon-like way, for you. four subunits, two each, of two different kinds of subunits, cell surface... <PAUSE WHILE WRITING ON BOARD> it's kind of like legs, sticking into, the cell. so you have, two of these shorter subunits, i'll point them out here. these extracellular domains the alpha, domains. these are the ones that actually bind the insulin itself. they are linked to the beta domain, which actually cross the cell, surface they actually cross into the intercellular space. these are the ones that have the catalytic function, of these receptors. this catalytic function is the phosphorylating of anything in its immediate vicinity that can be phosphorylated. so, this, receptor is itself an enzyme, with, kinase activity... <PAUSE WHILE WRITING ON BOARD> they are kinases. and specifically they are tyrosine... kinases. we may put phosphates, the group itself, on tyrosine residues of proteins that can take phosphates. so there it is it shows, this particular, cytosolic domain. let's move it up a little bit. and the key is, the binding of insulin, to these receptors, causes a huge, conformational change in the whole protein. what in fact happens is that these, these domains intercellular domains climb into each other. they have this huge shift, and they actually, i don't want oh i'll just write, climb into each other... and almost as if it were spontaneous, A-T-P in the cytosol was used, to phosphorylate the receptor itself. so, A-T-P at the expense of A-T-P... you put phosphate groups on tyrosine residues that are actually in this protein, this part of the protein chain. this is so-called autophosphorylation. so, they're climbing into each other A-T-P is in the cytosol, and phosphate is getting put on, the individual, domain. and once they're on one domain, that domain can phosphorylate another domain. and they, go back and forth and phosphorylate each other, whichever happens to be in the immediate area of A-T-P at that time. so they can do this continuous kind of phosphorylation then. they can put phosphorylation on, themselves, different tyrosine residues of the other domain, so they're, literally littered, with phosphates now decorating their intercellular domain. only on tyrosine residues. and what (happens) is of course these are membrane bond proteins, these receptors and they can slide along the membrane. and they do. and they slide along and guess what they run into every now and then. other receptors. other insulin receptors. so what'll happen is this guy'll slide next to another guy and whatta you think will happen to its intercellular domains? phosphorylation, until you have basically a pool, a pool of insulin receptors that are heavily phosphorylated in an intercellular (size.) and those phosphorylations, allow them, to run into other cytosolic proteins. cytosolic proteins like creatine kinase, for instance. they can become phosphorylated, and become active, or, become active in reverse, right? (cuz) you have to be careful about enzymes which way you're activating them. for instance you could run into, glycogen synthase, versus glycogen phosphorylase. it can, run into the enzymes that directly, phosphorylate synthase and phosphorylase. so you can imagine these guys, the, insulin, receptors' tyrosine kinase activity ultimately can be felt, to other cytosolic proteins that're floating around and happen to run into, because they have a specific domain that will run into, these, intercellular tyrosine (xx) domains. and what kinds of proteins do you think that go phosphorylate, or regulate? what is insulin a sign of? you have high glucose in your blood it's only sent out when you have high glucose. what is the signal? we've got plenty, don't make more. so what kinds of pathways will it affect? in the liver... what does it say? glycogen production. glycogen production. glycogen synthase is turned on, not directly it can't be. it can't be directly phosphorylated and turned on. so it has to be, affected at one level before that right? why would that? cuz glycogen synthase is in what form when it's phosphorylated? do you remember glycogen synthase? it's inactive, when it becomes phosphorylated. so presumably the insulin cascade is a protein, and activates a protein that does what, to glycogen synthase? takes the phosphate off, okay? what else does it tell the liver just for instance? okay does it need to do gluconeogenesis...? you've already got a lot of glucose in the blood. whatta you think? do you do you want your liver cells to be saying oh i wanna make even more glucose? no, turns on glycolysis. glycolysis is turned on, P-F-K-one, is stimulated. P-F-K-one is stimulated by what...? [SS: (xx) ] yup that's it. F-two-six visc- uh bisphosphate that's the signal molecule. that's the signal molecule that's gonna be affected. it's not directly affected by insulin itself, but its production is really affected by the cascade that happens inside. this is called a cascade, because it's one event that directly leads to another, event and directly leads on and on and on, until ultimately way down here, something gets phosphorylated, and the effect is seen. so it's an indirect multistep cascade. in the skeletal muscle whatta you think it would do there? you've got a lot of glucose that you can obviously import into the skeletal muscle. so, whatta you think? should you be breaking down your glycogen reserves? why bother, you've got plenty of glucose being transported in directly from the bloodstream. what should we be doing right here in the skeletal muscle? glycolysis, and the muscles are saying break down, break down... what else are you saying, in the skeletal muscle? storage, storage and you may have plenty, of glucose coming in, store this, make creatine phosphate, have a storage, of this high-energy molecule. because, you have plenty of energy to make the A-T-P, and you can directly transfer that en- energy into this high-energy, intermediate. so creatine kinase... is affected... store, creatine phosphate... store it it's a high-energy intermediate. you've got plenty of glucose coming in. that means you're doing plenty of glycolysis which means your making plenty of this A-T-P, it's there in high quantities. drive it into the production of something that can be stored in high quantities in the skeletal muscle. okay, insulin no signal is a signal, unless it can be turned off, okay? so how do you think insulin receptor can turn off its activity? for every receptor system that we look at, we have to consider how is it turned off. how is the receptor itself turned off, we've already talked about all of these things. only the cells that have an insulin receptor, will bind to the insulin. those are the only cells that will feel this cascade. the second messengers are what proteins are on the receiving end, of this, tyrosine kinase activity. what enzymes that's the same thing for this one because you don't have a very big cascade wait til we get to the next cascade, then you'll see the cascade. so how do we regulate it? no signal works unless there's a there's a method to turn it off. of course you could always just shut down insulin proju- production, right? so that's one level that you can shut this down. but, still there will be insulin circulating in the blood for quite some time after you've stopped producing it. the concentrations will go down very slowly there's a turnover rate, for these signal molecules. so you have to have a more direct, shut-off mechanism. any receptor will only be turned on in a pulse. that means ultimately this receptor has to be shut down directly by something that happens in the cell, and that happens, because, way down here protein after protein after protein ultimately, you phosphorylate a protein, that comes back around, and phosphorylates this enzyme this receptor this tyrosine kinase activity, on a seriate, or a threonine residue, and it shuts it off. okay? ultimately way down the line, phosphorylation of a protein... that specifically phosphorylates the receptor, and not at the tyrosine of course because that would just, serve to add more tyrosine kinase activity. but add a serinine(sic), or a threonine, and it's shutdown... that's one way it is known. when it is phosphorylated at a residue other than the tyrosine kinase activity, this receptor goes through another conformational change, the insulin is booted back out, and it is shut off. so it's an automatic thing it does it itself, autoregulation self-regulation of this, enzyme that is part of the receptor. another way, there has been some talk over the years years and years this has been talk, about the insulin receptor actually, becoming internalized. like the L-E-L receptor. because a lot of insulin receptor is found, in internal vesicles. so that's a great method that's how we shut down L-E-L receptor activity. whatta you do? bring it into the cell. it can no longer bind insulin, so, it's shut off. kay? those are the methods that, it can be turned off... in the most straightforward way. insulin receptor is one of the simplest, second messenger systems, or it contains one of the, simplest (xx) <NOISE DISTURBANCE WHILE RAISING BOARD>
S9: can i (ask a) (xx?) [S1: mhm ] um how c- can you dephosphorylate it? would that have any [S1: can you what? ] if you dephosphorylated the tyrosine that you originally phosphorylated, would that, would that turn it off? 
S1: you could but, nothing's ever gonna be shown to do [S9: (oh, okay) ] that to actually turn it off by taking phosphates off. presumably there's so much A-T-P accessible to it, and it has its own activity, that it could always replace it. you have to do something else, that is independent of the fact that the tyrosine keeps its phosphate. yes? 
S10: um, do all the phosphates that are attached to to the uh, tyrosines end up going to other proteins? 
S1: yes, [S10: all of 'em? ] well i mean there' s no way to really trace, one for one, but the fact is that the tyrosine kinase activity is heavily phosphorylated so the the protein itself, is heavily phosphorylated, and it is known to deliver phosphates to other proteins, whether all of them, on a one-for-one basis can be seen to do that. there's just no way of labeling that to find out. that's a good question, very good. okay, regulation at the point of what receptor you have, regulation at the point of what enzymes are turned on, let's look at another, a little bit more complicated, receptor, system. epinephrine. okay? hopefully by now, course you are writing, so that's a good sign. epinephrine, and, for that matter glucagon, belong to what is called <P :05> the adrenergic, system of receptors. specifically glucagon is considered, to to take part in what is called the beta-adrenergic receptor system. somatostatin, connects with receptors that are part of the alpha-two, adrenergic system, so it's a whole family... <WRITING ON BOARD THROUGHOUT NEXT 13:22 OF UTTERANCE> of this_ of receptors, that fall under this category. gotten the name of course, can you, think of where it got it? adrenaline. it's the receptor that was found to be directly involved in binding adrenaline which has since, changed its name to epinephrine, so epinephrine binds to these receptors, glucagon, binds to these receptors, course, adrenaline will bind to these receptors in what kinds of cells? mostly skeletal muscle. glucagon will bind to these kinds of receptors that are carried in what kinds of cells? mostly hepatocytes, so liver cells. somatostatin... binds to receptors from this family, and depending on exactly their composition their exact sequence their exact specificity, there're a whole bunch of different receptors that fall into this category. alpha-one, alpha-two, uh i think there's even an alpha-three but beta-one, beta, they had to number 'em. the beta-adrenergic receptors is, what we talk about for mostly epinephrine and glucagon. this is the main one, and it's what we're gonna follow first. the beta-adrenergic... receptor system. there's a great little description of what goes on on page seven hundred and sixty-three in your book if you wanna follow along. it is a receptor that is produced and put, on, biological membranes. it's kind of different than the insulin receptor, in its general structure. and in the fact that it has no, catalytic activity by itself. it does not specifically, catalyze reactions. not like the insulin receptor, which is, in its intercellular domain, an enzyme. this does not. this is a receptor that's mostly performing a structural role. it has a binding site... so this could be i think your book, has epinephrine binding to it. this could be epinephrine, or glucagon that basically go through the same series of events... just depends on which cell you're talking about, first level, of regulation. which cells you're talking about that would determine, which hormone, and in which receptor you have, and also which enzymes are inside and can be turned on and off. binding of... and then maybe by now you're getting hungry. are you getting hungry again? you can always come up for more bagels if you are getting hungry. but if you are getting hungry that's a sure sign that, glucagon has been sent out. and glucagon's saying to the cells, okay, wait a minute. you're in low blood sugar now. when your glucose in your bloodstream gets to a, below a certain point, a certain number of milligrams per deciliter or whatever, that can be monitored and are monitored by patients, certainly insulin pa- uh, diabetes patients with insulin problems. it gets below that point it drops below that threshold concentration what happens? glucagon's made glucagon's sent through the bloodstream and glucagon hits those cells, in the liver especially and especially quickly, they bind to their receptor, part of the beta-adrenergic family, and a huge conformational change occurs in this receptor. and this receptor has, kind of, um, in an intermolecular interaction kind of way, attached to it not covalently, but, just closely associated with it, a system of proteins... called, they belong to a family of genes called the RAS, family of genes, but they're called G-proteins. not for gee whiz, but instead, G-T-P-binding... and these proteins this whole family, comes from a whole family of RAS, genes, i'm sure you've probably heard that, name before, RAS, the RAS family of genes is a big oncogene family. a lot of cancers are a direct result of having, misproducts(sic). they are, mutants or, somehow they've been altered in this family of of uh proteins and they can cause horrible effects and uncontrolled cell division and uncontrolled cell growth. tumor, promotion right there <POINTS TO BOARD>, from this family. and it's, the G-protein family. and they're involved in many cellular processes, this is only one of 'em. they're involved in huge numbers, the RAS the RAB, the RAM the all of them that are directly related. and their whole job, is to bind G-T-P, and hydrolyze it. but, what it's interacting with, while it's doing its job, is causing conformational changes in those proteins that it's interacting with, and turning on and off their, activity. so its whole job is to bind G-T-P, and move around and hit and run into other proteins. there're three subunits, on the_ on a typical G-T-P-binding protein. the alpha subunit is the catalytic, subunit. the beta and gamma, are basically regulatory. so, when G-D-P is bound, to this alpha subunit, what happens is it it has this confirmation, that puts it, in close proximity to actually cozying up to its beta and gamma subunits. and the gamma subunit of this protein of this class of proteins, actually adheres very nicely, to the beta-adrenergic, receptor. okay so these are in close proximity. they are closely, they're not covalently bound, but they are, in close, attachment... noncovalently. so when, G-D-P is bound... this threesome, is tightly, adherent, to this receptor. when the receptor binds to its hormone, what happens is, there's a shuffle. G-T-P, boots out the G-D-P, and the binding of this alpha subunit to G-T-P, causes such a huge conformational change in that subunit, that it sails away, from the alpha_ from the beta and gamma subunits. it actually goes, sailing, across the intramembrane (leaf,) alright? cuz this is a bilayer. so... this alpha subunit is actually kind of cozying up to that, pho- (phospholic) that heads along this bilayer. and it gets sent out. it binds G-T-P, and it gets sent out and it's skating, kind of skating along, the membrane, at its intercellular surface. and it skates along and remember this is a cell, so this is three dimensional. this is skating in all directions, and it's in this G-T-P-bound stage, so it's skating until it runs into a protein that in this confirmation, it actually attaches to not covalently but intermolecularly. and the protein that it attaches to is also a membrane-bound protein, right here. your book calls it adenylate cyclase, other books call it adenylyl. so don't be worried if you're seeing different, spellings... adenylate cyclase or adenylyl, cyclase. and what does this guy do? so, even though it looks like a kind of stoichiometric thing here where one epinephrine's bound to one receptor and one G-T-P protein is floating along to one aden- adenylyl cyclase. it's not, remember this is happening in three dimensions. so it's not like they're in an electron transport chamber, and one feeds directly to the other. this is skating all over, and it's seeing as many of these adenylate cyclases as it can, and when it hits it... this is the alpha subunit, it turns on the adenylate cyclase. and the adenylate cyclase has a cytosolic, domain that does a very important, catalytic event. it makes a second messenger. it, takes up A-T-P in the intercellular domain, right here and turns it into cyclic A-M-P... pyrophosphate is the least, so you're going from a triphosphate molecule, to a monophosphate molecule. and this monophosphate is actually, linked... in a three-prime to five-prime prime way okay, so it is a cyclic, three-prime, there's your three carbon, to five-prime, phosphorylated molecule. we call it, cyclic A-M-P. and as long as that, alpha subunit binds to the adenylate cyclase, it's catalytically active. and it's chugging out C-M-P left and right. and cyclic A-M-P is, what we call a second messenger... it was hard to define second messengers in the insulin, cascade. because so many different proteins were directly affected by the insulin receptor itself. but in this case, the receptor had no activity, and this, concentration of this internal, uh if you will, metabolite increases dramatically and it has cellular effects. so that the second messenger its levels... increase, as a direct result of adenylate cyclase, which is only turned on, by the alpha subunit, of the G-T-P-binding protein... and which is now ultimately leading back to, the receptor-binding, event... direct effects, indirect effects... directly it's turned on by this enzyme making it. but that enzyme is turned on by, G-T-P-binding protein, attaching to it. and that's turned on by, the hormone binding to the receptor. kay? so it keeps going. and cyclic A-M-P is made in a lot, in huge concentration, a lot of events. so it's often thought that second messenger systems and these cascades that directly result, no matter how many steps, are in between, from hormonal binding events, are, giving you what what the cell would call what you would call ampli- amplification, of your signal... you get, an amplified signal, with every second messenger, that is turned on, as a result of this hormonal binding event. you don't just make one cyclic A-M-P for every one hormone-binding. you make, seven billion, cyclic A-M-P, for every one, hormone-binding event. so your signal is now, seven million times stronger, within the cell. <WRITING> with every, second, messenger. </WRITING> okay? so beyond that_ yep?
S11: um, is the glucagon that binds to the protein, is that what stimulates the G-T-P to, attach to the
S1: yes [S11: okay ] it's what makes the first placement. it binds with it it its beta and gamma subunit only when the G-D-P, is in that pocket. when G-T-P, boots out the G-D-P, so basically you're gi- you're you're losing your G-D-P that was bound, and you're putting in place of it a G-T-P, a high-energy molecule, that's what sends it sliding along. so whatta you think sends it sliding back? what's it's whole job...? it hydrolyzes G-T-P. that's its whole job. so, as long as it still has G-T-P bound, it will be in search of adenylate cyclase, in every direction. and, transiently bind to it, and turn it on, until it, makes a lot of cyclic A-M-P and then it'll, bounce off of that and go to another one maybe. but, at a certain point it's actually going to do its catalytic event and what's it going to do? it's going to hydrolyze that terminal phosphate on the G-T-P and what will then be in its place? G-D-P. and what_ where will it go? and that's the main way that this has a shut-off switch. it has only G-T-P bound for as long as it can keep it in its active site before it actually does its job, hydrolyzes the, the third phosphate, and has G-D-P and then it has a different conformation that makes it bounce back into, the beta and gamma subunits. so, let's actually write that. <WRITING> the regulation, for these, types of receptors at first level, the G-T-P-ase activity... of the G-protein... </WRITING> mkay? that's the first way that that's regulated. there's another enzyme, actually that's affected by other, hormonal signals that we'll get to tomorrow, that actually breaks down cyclic A-M-P. this enzyme that takes <WRITING ON BOARD THROUGHOUT NEXT 12:59 OF UTTERANCE> cyclic A-M-P, and turns it just into A-M-P is another way, in which, regulation happens. so it's no longer at the point of the receptor anymore that you're regulating this cascade. you're actually shutting down, and reversing, production of the signal molecule. this enzyme that does this is called phosphodiesterase... and we get into this in a big way... tomorrow when we talk about (another) cascade that directly turns this enzyme on. okay? how many of you have heard fro- of cholera toxin? cholera toxin results in, i- it (is a) direct result of, an infection that causes, the alpha subunit to be what is called A-D-P ribosylated, it actually gets, in place of the G-T-P, that's put on it you get a, covalent attachment of an A-D-P ribose, which effectively turns this alpha subunit on constantly. so cholera toxin is a direct result, or directly results in, turning this on constantly, making all of your cells, that are affected by this hormonal cascade, turned on constantly, and you end up with a whole series of symptoms that are a direct result of this, <POINTS TO BOARD> guy being permanently, affixed in its, in its on position. and i think that whooping cough, pertussis toxin, affects the inhibitory subunit which we're gonna talk about in a minute. there's a there's another, somatostatin, it's a receptor that has instead of an active, G- what we call G-S-protein, a stimulatory G-protein this one, it ha- it actually, activates an inhibitory protein, a G-I. let's (look) up here second, okay what happened to this one? cyclic A-M-P levels increase in the cell, let's follow this to its completion and then we'll talk about somatostatin and the others. what happens? inside the cell cyclic A-M-P is, in huge concentration. and it, specifically binds, to proteins. and those specific proteins, that bind it quickly, there's one, specific one. that is called, protein kinase, do you know? (kay?) it's a cyclic A-M-P-dependent protein kinase, that's been called, protein kinase A... <WRITING> dependent, protein, kinase, P-K-A. </WRITING> so what happens? there are two regulatory subunits on this protein kinase and two catalytic subunits on the protein kinase... regulatory regulatory, each regulatory subunit so how many if i did an S-P-S page, how many protein bands would show, if you have two identical, regulatory subunits and two identical, catalytic subunits, on this protein? [SS: two ] only two. and each would be a duplicate but you can't tell that on a gel. cyclic A-M-P binds to that regulatory subunit, in, two places on each regulatory mole- protein chain... and get this, what happens is when all of the cyclic A-M-P spots are filled, the regulatory subunits let the catalytic subunits go. so, this guy's completely inactive. these catalytic subunits are completely inactive when they are tied up, with the regulatory subunits. but binding of the regulatory subunits to the cyclic A-M-P, releases the catalytic subunits. and they are now active protein kinase A and so whatta you think protein kinase A does? well it, phosphorylates a lot of proteins, that's why it's called protein kinase. protein kinase A phosphorylates a ton of proteins, one of which is phosphorylase B kinase. do you remember phosphorylase B kinase? so what'll you think it_ what'll_ whatta you think that effect will be? if it phosphorylates, phosphorylase B kinase, and activates it, then phosphorylase B kinase will phosphorylate, phosphorylase B, and what will it do? activate phosphorylase B, and glycogen breakdown will begin... <WRITING> phosphorylase, B kinase </WRITING> and ultimately, glycogen breakdown, begins. what else does it do? oddly enough, in the liver, glucagon, stimulates fructose-two-six-bisphosphophatase. why? as that signal molecule. when glucagon is high, the (physio) molecule is diminished. two-six-bisphosphate is the activator for glycolysis. and in the liver, you don't want glycolysis going on. in the liver your role is in producing glucose. you've got your glucose as a signal. so the liver is saying, okay, i'm in trouble, i wanna shut down glycolysis, which means my P-F-K-one has to be inhibited. so i'm going to lower, effectively lower the concentration of fructose-two-six-bisphosphate... that signal molecule that does that. and that means there's less activation of P-F-K-one. don't get lost in the cycles here they're, they're very complicated so, just get the major input. what's the problem? glucagon, low sugar. you're getting hungry, you're for glucose production. the liver has to produce glucose and the muscle, has to make, itself use the glucose that the liver is sending out. so that's, the cascade. now each, activation we get, doesn't just result in one other activation event. so, every time you have this release, you have these enzymes active for as long as they, can do their job before cyclic A-M-P is released, and lowered enough in concentration for these to swing back to the regulatory subunits. so again you have, amplification... it doesn't just do one phosphorylation event. it does thousands because it's active for that amount of time. same with this. when it activates phosphorylase B kinase, it doesn't just mean phosphorylase B kinase turns around and does one activation, on glycogen, pho- phosphorylase. it does lots of it so you get amplification again. now it seems to me, this hormonal cascade, runs circles, around this one. do you see why? i mean this guy has almost virtually no second messengers. you know, none that you can identify as being consistent in every cell, it's got its own activity. it's rather rather simple and beautiful in its, in its method. but this guy, has an enormous number of proteins being turned on, and being affected by other cascades. it's a whopping cascade. it is a huge, cascade of events, and with every event, in this cascade, there has to be regulation. with every event. just like, glycolysis you couldn't just have one bypass at the front. you have to be able to re- regulate every step along the way. and to some extent you can. at least the irreversible ones. so for this one regulation has to happen at every event. so, at the same time as you have self-regulation by the G-T-P-ase activity, you have, another enzyme involved cutting back this, signal. you have other enzymes involved in regulating this whole, set, of protein phosphorylations. so you have the, counteractivity, of numerous enzymes. along the way. and this, kind of, competition, if you will, between the activities of different enzymes whether it's going to go, yes to the closed cascade or, stop the cascade, that's called cross-talk cross-talk, is, different cascades affecting, other cascades. so there is cross-talk between the insulin signal, and the glucagon signal. there is. if the cell has an insulin receptor and a glucagon receptor, insulin, the whole cascade, effectively turned on by insulin-binding, will shut down, this guy's cascade. and not just that one event, all the way along. there are proteins being phosphorylated here by this binding event, that will directly reverse, these steps. another cascade that we're gonna get to tomorrow is the vasopressin, cascade, a whole different set of enzymes. and it also cross-talks at all these different places. and there are specific places where they will overlap and they will shut each other off. so one other way that it's regulated is, you have to think, again, you have to back off and get a whole cellular picture. this doesn't do justice to the fact, that this is going on in every direction. okay you have to imagine the whole surface has these kinds of little ships, that're all these G-proteins, these alpha subunits, these stimulatory... what're called G-S-proteins because they stimulate adenlyate cyclase... they're floating, and they're hitting as many adenylate cyclases as they can, in the same cells, there are somatostatin, receptors. and somatostatin is involved in, a lot of the cross-talk between these two cascades. it's moderating the insulin versus epinephrine and glucagon signal. somatostatin has the same kind of, receptors. they belong to the same adrenergic family. but they have a different overall structure, and they're called alpha, instead of beta, adrenergic receptors. and instead of using G-proteins, when somatostatin binds to its receptor, it sends off, G-inhibitory proteins... um, <WRITING> somatostatin </WRITING> has an alpha receptor, and it binds to an inhibitory, G-protein. so, somatostatin effectively sends off these inhibitory proteins, and where do they bind? to the adenylate cyclase. so if somatostatin is released because these two signals are going on at once, what's going to happen? it's going to shut down this pathway here. <POINTS TO BOARD> because those inhibitory G-protein alpha subunits, are gonna be sailing forth, and what're they gonna hit? <POINTS TO BOARD> these. the same things that the stimulatory ones are hitting on all sides of the cell. they're gonna float around, and they're gonna shut it down. so that's one way that cross-talk happens. you have an inhibitory subunit instead of a stimulatory one. so it's in effect blocking this guy's activity. because, presumably both signals are being, are being considered at once by the cell. is this pretty frightening so far? [SS: yes ] it is. i knew it would be. you see i'm so used to_ that's why i fed you these before. there's always a reason. so keep eating, keep eating and you'll be able to follow along. we'll we'll pick up with it tomorrow, and please come down and have more bagels cuz i really don't wanna carry 'em away with me. come and eat bagels, take 'em for the road, it's noon it's lunchtime.
{END OF TRANSCRIPT}

