(Small) Molecules of Life

by David Siegel

An Economic View on Human Biochemistry
“The human body’s chemical value is just about a couple of bucks.” - a frequently read line and a great understatement. While it is indeed true that the value of the chemical elements found in a human body, mainly oxygen, carbon, hydrogen, nitrogen, calcium and phosphorus in their pure form is not more than a couple of dollars or euros, this is not what we are actually made of. Our bodies are rather incredibly rich agglomerations of biomolecules, made from those elements, ranging from simple compounds like water to huge biopolymers like DNA or collagen, the major structural protein in our skin, hair, muscles or tendons. In this chemical treasure trove the pure elements are barely represented - neither by mass nor by number.

Biomolecules, as well as their mixtures and structures (think of organs, eggs, bone-marrow or blood) fulfil key functions in human biology and thus gain their worth. A fact which needs to be acknowledged when attempting the calculation of a body’s “chemical value”. It is obvious that such calculations are sketchy as human body parts are usually not sold freely - at least not on legal marketplaces. Still, the magazine Wired attempted an estimate in its February 2011 issue [1] and arrived at several million US dollars for the components of a human body, sold individually.

The difference between the two prices, “a couple of bucks” and several million dollars, one for the plain elements, the other for molecules and structures thereof, is a (sketchy) measure of the value of chemical information inherent to our bodies’ biomolecules, which is, again, a measure for the incredible degree of optimization achieved by evolutionary processes. In his 1977 Lindau lecture, German Nobel Laureate Manfred Eigen gave the audience a straightforward yet impressive introduction on the power of evolution with respect to the generation of such (useful) biochemical information:

Manfred Eigen on the Evolution of Biological Information (in German)
(00:01:53 - 00:04:48)

Structuring the Biochemical Treasure Trove
The molecular information generated in several billion years of evolution is vast. Even today, no one would dare to claim to understand all the chemistry going on in a human body – even keeping track of the tens of thousands of molecules we do know about proves difficult enough. Still, there are ways of classifying our biomolecules which can make life a lot easier.

First off, there are the biopolymers, chain-like molecules built from recurring, well-defined links. Our DNA is a biopolymer built from a set of four nucleobases. Similarly, our proteins are biopolymers built from 21 amino acids. Biopolymers are characterized by an easily comprehensible, linear construction principle, which – at the same time – allows for a theoretically limitless number of molecules. The only restricting factor is the achievable chain length. Hence, DNA and most proteins are what biochemists consider “large” molecules.

Next to the large biopolymers, there are non-polymeric molecules most of which qualify as “small”. By convention, this means that their atomic mass is below 900 Dalton, the mass of 900 hydrogen atoms, 56 oxygen atoms or 50 water molecules. In our bodies, small molecules take over a variety of functions – nutrients like carbohydrates and lipids partake in metabolism and thus act as a source of energy and chemical building blocks required for growth and development. Neurotransmitters, hormones and pheromones are involved in signal transduction. And vitamins and cofactors support enzymes in conducting essential biochemical reactions, to name only the most prominent examples.

Small molecules may enter the human body via inhalation, absorption through the skin and most importantly: via food. Once taken up, they are subject to a variety of metabolic processes. Small molecule nutrients are broken down to gain energy and chemical building blocks or converted to other compounds, which can be stored or transported more efficiently. Potentially toxic substances are equally degraded or chemically modified to facilitate excretion. And, most compounds are, to a lower extent, subject to random chemical reactions, which are unintended by the body’s biochemical machinery. Taken together, these processes account for the huge number of small molecules found in our bodies. Today, we know of more than 40.000 such compounds [2]. Reasons enough to put DNA, RNA and the proteins on the side and dive into the world of our (small) molecules of life.

Dealing with Macronutrients: Central Carbon and Energy Metabolism
Next to the proteins, small molecule macronutrients like carbohydrates and lipids are essential for our survival. As opposed to micronutrients, they are required in rather large quantities and can be considered raw materials required both as “fuels” and for the generation of chemical building blocks. Our body is equipped with a highly optimized biochemical machinery, allowing for an incredibly efficient utilization of such macronutrients. Humans metabolize as much as 93 to 95 % of the macronutrients contained in a typical mixed meal [5].

In the world of macronutrients, everything revolves around carbon. To obtain energy, our body converts energy-rich, carbon-based macronutrients to energy-poor, stable molecules, most of all carbon dioxide. Details on the chemical journey of carbon, from macronutrient to carbon dioxide and the associated Nobel Prizes, can be found in the Mediatheque’s Topic Cluster on Carbon. Notably, these so-called oxidation processes do not occur in a single step but proceed through biochemical pathways like glycolysis or the citric acid cycle, involving a wide range of intermediate compounds. Taken together, these small molecules are referred to as central carbon metabolites or energy metabolites. They are found in practically every cell.

However, our bodies do not stop at just degrading macronutrients. To a certain degree, they also interconvert them. One of these interconversion processes leads to an effect dreaded by many: weight gain, i.e. the accumulation of fatty acid triglycerides in fat cells. Fat pads are in essence reservoirs of small molecules used for energy storage. In this context, it is a common misconception that the build-up of fat can be avoided by a fat-free diet. Our bodies possess the ability to biosynthesize fats from very small building blocks, containing only two carbon atoms. The body can source these building blocks from carbohydrates and proteins and thus produce fat de novo. At the 1966 Lindau meeting, 1964 Nobel Laureate Feodor Lynen, who had been honoured together with Konrad Bloch for his contributions to the elucidation of cholesterol and fatty acid metabolism, explained to the audience why humans are largely independent of dietary fat:

Feodor Lynen on Fatty Acid Biosynthesis (in German)
(00:00:15 - 00:04:11)

Macronutrients vs. Micronutrients
Towards the end, Lynen mentions that certain fatty acids cannot be biosynthesized and hence need to be taken up with the diet in quantities comparable to those applicable to vitamins. These two fatty acids, α-linolenic acid and linoleic acid, belong to the group of micronutrients. Micronutrients are essential, but not required for energy generation. They rather support vital metabolic reactions, either directly, as enzyme cofactors, or indirectly, as building blocks for other biomolecules.

Minerals, trace elements (e.g. iodine) and vitamins complement the spectrum of micronutrients. In the realm of the Nobel Prizes, vitamins in particular play an outstanding role. No other group of small molecules has motivated more Nobel Prizes. This may be explained by considering that the concept of micronutrients in general and vitamins in particular was not well established until the first half of the 20th century. The story of vitamin discovery hence coincides with the story of the Nobel Prize, which was first awarded in 1901. Whereas (with few exceptions) the major macronutrients were isolated before Nobel Prizes were awarded, vitamin researchers just were in the right spot at the right time.


Vitamins

Discovery and Nobel Prizes
All 13 vitamins we know of today (A, B1, B2, B3, B5, B6, B7, B9, B12, C, D, E, K) were discovered in a period of about three decades, from 1913 to 1941. With the Nobel Prizes being awarded since 1901, the chances of receiving an award for vitamin research could not have been better and, in fact, as many as eight Nobel Laureates owe their Prizes directly to work on the discovery, isolation or structural characterization of novel vitamins [3]. Several additional Prizes were given for work involving vitamins in part.

However, it was only in 1929 when the Karolinska Institutet decided to honour vitamin researchers. After all, the concept of micronutrients and vitamins was still new, even the term “vitamin” had only been coined in 1912 (based on the false assumption that all vitamins feature an amine group, vitamin was short for “vital amines”). And by no means were vitamin pills available over the counter like they are today.

Still, once the importance of vitamins became apparent, a true wave of vitamin Nobel Prizes ensued. It began with the 1929 Prize in Physiology or Medicine, which went to Frederick Hopkins and Christiaan Eijkman for their work towards the discovery of the vitamins A and B1, respectively. These two vitamins were the first discovered and the first connected to a Nobel Prize. Interestingly, neither Eijkman nor Hopkins had isolated the pure vitamins or determined their structure. They merely pointed out that certain foods (milk in the case of vitamin A and brown rice in the case of vitamin B1) have the potential to cure certain conditions arising from an unbalanced diet. Others later identified the actual compounds responsible.

In 1937, both, the Prize in Physiology or Medicine and the Prize in Chemistry went to vitamin research. And both Prizes were motivated essentially by one compound: ascorbic acid or vitamin C. Albert von Szent-Györgyi Nagyrápolt was rewarded in Physiology or Medicine for its isolation, whereas Walter Haworth received one of the Chemistry Prizes for synthesizing the vitamin (in 1934). Haworth’s co-recipient, Paul Karrer, was acknowledged for his work on the vitamins A, E and the B vitamins.

Ascorbic acid, the lack of which caused the famous scurvy amongst sailors, was long sought after. It had been known for quite a while that foodstuffs like lemon juice can prevent scurvy. However, that a single small molecule compound was responsible for this effect could only be established in the 1930s. In his 1969 Lindau lecture, the Hungarian Szent-Györgyi described how he “accidentally” isolated vitamin C while wondering about why certain fruits turn brown when damaged, while others do not:

Albert von Szent-Györgyi (1969) - Molecules, Electrons and Biology (German Presentation)

Count Bernadotte, Colleagues ... (inaudible), thank you. Ladies and Gentleman, As you know, biological thinking today is permeated by the influence of the molecular theory. And according to this theory, the living system is composed of closed particles with closed electron shells, molecules. And heat agitation causes these molecules to oscillate and collide on occasion. I have never really been able to believe that this is the sole source of the wonderful intricacy and adaptability and fittingness of living nature, and here in Lindau I asked myself whether our situation is not analogous to a man observing the meeting of Nobel Prize laureates in Lindau from a height of 10,000 meters. If this person were asked: "What are Nobel Prize laureates?", he would say: Sometimes they collide, then they part again, and that's all there is to it". I have never really been able to believe this, and I would say that the main outcome of my long years of research of more than 50 years is a profound respect and admiration for the marvellous complexity of living nature. That these molecules are not really as isolated and not really as closed as the molecular theory would have us believe, the first indication was discovered by Weiss in England in 1942. He found that if a strong oxidising agent and a strong reduction agent are combined they form a complex. And this complex then develops a dipole moment, meaning that the molecule has a positive and a negative charge. And straight away he understood rightly that this could only happen through an electron transferring from the one molecule, from the reductant to the oxidiser. For as we know, oxidising agents have a strong desire to accept electrons and reduction agents are keen to release electrons or hydrogen atoms. So, this all adds up well. He interpreted the whole sequence – may I have the first slide – correctly at first glance, that the electron, with the one molecule releasing an electron which becomes the donor, right? The other accepts it, thereby becoming the acceptor, and these are then given the abbreviations D or A. So as Weiss accurately reports, in the first stage they form a complex, a stable configuration held by normal forces. Then an electron is transferred within the complex. An electron passes to the acceptor. As you know, all molecules are composed of electrons which always come in pairs. There are always the two together. They come in pairs, which means that an electron spins off from its pair and transfers to the acceptor, which then becomes negatively charged. No one has ever seen this happen – it's something we can't see. All we have is a good dipole measurement from measuring the dipole moment which is awfully difficult and very complicated, but we still can't see anything. If, however, the conditions are very favourable – extremely favourable – including the relative energy, the pair may break apart and become two independent particles, an acceptor with a negative charge and a donor with a positive charge, which is what we now call a free radical. So then we have a free radical. And this, which is something we can already prove, may emit a sign or a signal in an electron spin resonance machine. The electron spin, by which I mean the machine itself, has 1,000 buttons and fills a whole room, and as you know, one of the laws is the smaller particle the larger the machine needed for proof. So the electron spin resonance spectroscope can detect when an electron has separated from its pair and is sitting there by itself. So, what's the best way of imagining this - can I have the next slide. Here - as you know, the electrons in molecules or atoms form a sort of cloud and the cloud has a certain localisation called an orbital, and these orbitals have different energy levels. This slide shows occupied orbitals, there are, of course, occupied and empty orbitals. At this point, you will say that it's not empty at all. What we have here is a physical reality, but there is nothing sitting on it, sort of like an empty chair. So here are the occupied orbitals, the ones with the thick line, and those are the thin ones, which serves to indicate what is an oxidising agent and what a reduction agent. This means that an electron is in a very high orbital, at a very high level, with a strong propensity to transfer. If the acceptor, meaning the oxidising agent, has an empty chair, namely at a low level, the electron can go and sit on it. This then generates the energy and the two charges, the positive charge This is then the ground state, with no excitation. Only the previous carrier energised. This is the transition to the ground state or, to put it another way, strong charge transfer. Another type of charge transfer was subsequently discovered and researched in detail, in particular molecules with spin-up electrons. Could I have the next slide please? Naturally, an electron could never spin to a higher level by itself. It lacks the energy to do this. If, however, it happens that an electron is energised by a photon, by light energy, this energy can send it to a higher level, which is then charge transfer in an excited state, isn't it. Life's most important chemical reaction is of this kind, as exemplified by the excitation of the chlorophyll molecule in plants. The electron is excited by light and transferred to a higher level. So, what usually happens is that the electron naturally falls back again, the cloud is distributed between the two molecules, and the electron falls back. But life has learnt to grab hold of the electron here with a metal atom, with an iron atom, and then it has a little energy, and this is the energy which sustains you, which sustains us all, from this energy supplied by the photon. So, to summarise, the next slide please. So, there are two electron transfers, where an electron jumps to a higher level and where it falls back, which is the ground state. Now a question for the biologists: What can biology make of this? Nothing whatsoever, for the simple reason that there is no light in our bodies. Consequently, when we talk about animal physiology, there is no light in the body And there is no strong oxidising agent in the body either, as we would not be able to tolerate it. They would kill us, the strong oxidisers. Which is why there are none. As a result, there would be no chance of a charge transfer. Charge transfer could, however, be extremely significant. Indeed, if there were to be just one, even in this middle ground, where the electrons, where the substances are neither powerful oxidisers nor strong reducers. If they could display just one charge transfer. Because our whole body is constructed on substances, which are not strong oxidising agents. So my main question to put to the experts is now: whether, in this middle ground, which forms the basis for the whole body, a charge transfer would be possible. But now comes the difficult part: How could you prove this? In this region, indeed, waiting for an electron, for the whole electron cloud to transfer to another molecule would be an impossible option. It would merely spread, the electron cloud. That which we observe in electrons, like a cloud. The cloud would merely distribute itself between the two molecules, the donor and the acceptor, and in this state of dissipation, getting an electron spin resonance spectrum and an indication in this machine is not yet possible. So is there a way forward? Yes, there is, with a small trick. Small tricks are indispensable in science, because if one needs especially favourable conditions for a whole electron to transfer, yes - the attempt should then be made to create such favourable conditions, and then to observe whether the electron wants to pass over with the substances that are actually present in our bodies. After all, we have all sorts of substances inside us, proteins and amino acids and, taking all this, and having created particularly favourable conditions by introducing a strong acceptor in a particularly good solvent – there are special properties which one anticipates – and we then put the whole lot in the electron spin resonance machine and what we get is a wonderful signal. Anyone who has worked with an electron spin resonance machine knows how marvellous this signal is. There are intervals which we can use to calculate everything. This is a wonderful signal. And we found that hydrogen atoms and oxygen atom can donate electrons. So, this is really astonishing. As you know, electrons are always bound in most compounds. However, hydrogen and oxygen as well as sulphur and similar atoms have electrons, two electrons, always pairs of electrons that are not bound, chemically bound. These are called a lone pair of electrons, the French always say, "cherchez la femme", so in French this would be " unmarried electrons", "électrons célibataires". So, one of these unmarried electrons can transfer perfectly easily. Both from hydrogen, especially, and from oxygen. As well as sulphur. What we get is a completely new idea about life. And about living organisms. Also about the relation between molecules, and about everything that these delicate reactions could signify. So, we have a kind of cloud, an electron cloud, which disperses across two atoms. This is a spectacularly delicate connection where anything and everything could happen with the electrons. This includes the completely unexpected. And up until now, we have always believed that carbon is present, with its four valances and large molecules, which is correct. However, oxygen and hydrogen are only present to create acid and alkaline valances and possibly hydrogen compounds and so on, otherwise they don't have a purpose. But now we come to a new idea. Perhaps this is completely wrong. And perhaps this is the essence or one of the intrinsic factors of life, these electron clouds. And each hydrogen atom and oxygen atom, along with carbon and sulphur atoms, are wonderful donors with their unmarried electrons. Yes, but such a donor only makes sense if there is an acceptor. When a girl runs around finds no one to take her in, she makes no progress. Yes, acceptors are also needed. So, what are acceptors? No atom can be an acceptor. There is, however, a compound, the C-O bond, or every double bond, every double bond is a good acceptor. Oxygen is not alone in not being an acceptor. Carbon is not an acceptor but the electron clouds, present as an occupied electron cloud always in a double bond An empty one, which is also a good acceptor. But this makes the whole thing really exciting. And this is why: - Can I have the next slide please. This is a peptide bond. As you know, Thyrell discovered, or this was known already, that all proteins are made up of a huge number of amino acids, and two amino acids join with a peptide bond. There are therefore innumerable peptide bonds. Almost only one less, as many bonds as there are amino acids in one molecule. So, here is a peptide bond and there you can see what is so exciting, that's an acceptor, this double bond, a strong acceptor group, and here is a strong donor group. So, each bond has a donor, which means the possibility of a really large number of these change transfers This gives us a completely new possibility, doesn't it, for it is then possible that these clouds, here we say a peptide chain – next slide please. These are peptide chains here, which I have simply labelled as peptide chains. And when this oxygen generates a charge transfer with the next peptide chain, and this in turn with the one after, then the whole protein molecule or two molecules touching one another here join to make up an electron cloud. And this gives rise to quite different ideas about the real significance of a protein. Indeed, this leads to infinitely new things, I mean that it is only a fanciful notion at the moment, but a beautiful fanciful notion. So, those would be the possibilities, and each such charge transfer is a bond, evolving into a very large number of bonds, with weak bonds appearing, between protein chains or various molecules. So far, so good, but how to prove it? Proving it is not possible, unfortunately that doesn’t work. Simply because these transfers, meaning the cloud, do not sit on an atom but are dispersed, and therefore do not emit an electron spin signal. So what we can do right now is to investigate simple proteins and ask: Therefore, assuming we have the picture right, there must be very many weak bonds in the peptide chains and between the molecules, a veritable multitude. And a system where the components are held together by weak bonds, many weak bonds, has properties that are quite different from a system with few strong bonds. May I have the next slide please. Here I have roughly outlined the whole thing. Two molecule surfaces bound together by a strong bond, and here two molecule surfaces bound together by many very weak bonds. So what is the difference between the two? This is quite a delicate matter. To separate the two molecules I need enough energy to break the bond, let's say around 15 calories. Indeed, if I have a few weak bonds here, say 15 of them, I need the equivalent of the same energy as before if I want to proceed in this direction. But not if I come from the side. If I start at the side, here for instance, then I only need to break one bond at the moment if I start here. This is only one calorie. Then we move to the next bond, the second calorie, and then the third, so with a minimal amount of energy, namely only one calorie, I can break up the whole thing. And this has very interesting characteristics. If I break one bond, the next will be predisposed to break. It is too weak to hold the whole thing. It breaks as well, and the whole process is self-perpetuating. And this also works in reverse: if I create a bond, the next will form more easily. As you can see, the whole thing can continue to move along. It makes up a system, an "all-or-nothing" system, where either everything happens or nothing at all. We see systems of this kind in everyday life, don't we. Take a zipper, something you are familiar with, right? The zipper on a pair of men's trousers, such a zipper. A zipper is such an "all or nothing", you know it can only be unzipped from one end if you want to open it. And the it is zipped upwards. Closed from the other if you want to reverse the process. And, sometimes to our chagrin, it parts way in the middle, which opens the whole thing up and causes great inconvenience. There are various kinds of all-or-nothing stories, which can have the same effect. And what’s more - the characteristics that emerge can be completely weird. If I have two particles which have this weak energy on the surface binding them, they can rotate freely around each other without using energy. Because on the one side I then break the bonds and, on the other, I create bonds. This activity therefore costs me nothing. They can move around without any energy. They behave, in other words, like a liquid. At the same time, however, they are always held together at one point, which means they behave like solid matter. This is really strange, they are both solid matter and liquid, in one state and in the other. And living tissue has these properties. Wonderful. Take the tissue which is most easily accessible, skin, here is my skin, and this is the point: it is neither solid In my view, very robust, my skin would do any boot proud. At the same time, however, it is fluid, I can pull on it. And then it returns to its original state, to put it another way, it is fluid in its movement. This is the way it is, and then we progress to something very important, if I cut myself, these are cells which have remained undisturbed for 75 years and they are bound together in a solid configuration, all very firm in one respect. But when I cut myself, the picture then changes completely. The cells that were formerly strongly bonded together part ways, seep in a fluid movement into the wound and disperse. And in order to disperse a cell must be fluid, because otherwise it would not be able to make this transformation. So, all of a sudden, the whole thing takes on liquid form. And with anything to do with wounds, we need to be careful, very fluid, not to be touched. And as soon as the whole thing has sealed up, it fills out and a scar forms which is even more durable than before. As you see, there are these huge changes, these "all or nothings", these all-or-nothing changes, either into the one or the other state, either a fluid, active state or firm, static state. Consequently, we need to place a new demand on the system. Namely, if this is so, in this scenario, that there are ruptures in the wound, suddenly all these bonds are ruptured and it liquefies. Consequently, these bonds must be of a kind that can be easily and suddenly split. I cannot, for each bond, something special So the bond is that way. Electron charge transfers behave in a similar way. Because if I introduce strong donors, meaning electrons, into the system, the electrons will ... let's say, here is a donor and an acceptor which form a bond, the new acceptors which I introduce will compete with these donors and have a splitting effect. So by changing the electron voltage, therefore to speak, I can cause the whole thing to break up. Or to change if I introduce an acceptor to bind all these free electrons or strong, extremely active electrons, as the chemists would be sure to say, about the low ionisation potential, then the activity needs to cease, which then achieves a stable state. So this all makes sense. And then I look around to see if nature actually applies this principle. This acceptor-donor principle in its manipulations, and I have always been interested in plant oxidisation systems, and the first system which I worked on was the polyphenol oxidase system in plants. Yes, I need to add something. If a living system is able to induce a stable state through an electron acceptor which is introduced, I can use an electron acceptor to do the same, to eliminate all active electrons. Let's take an acceptor, and the acceptor I introduce must be a lot stronger than the acceptor lasts, otherwise it won't be able to compete. So how can one – I know only one acceptor and that is the CO bond, in actual fact a double bond, although there is also CC bond as well as NN – increase the activity of this bond? The answer is that I can't. So what can be done? How about taking two. That's simple. Then it will be twice as strong, won't it? So, if we take two, and if this does not suffice, I can take two COs, which means I can then count another double bond, a jointly conjugated electron system, which then becomes extremely strong. So, what is the strongest acceptor for regulation? Next slide please. The strongest acceptor would then be this one. In other words, a simple aromatic ring, and which is then immensely strong. It's the strongest, so strong a regulation that it kills everything. Killing is also a regulation where irreversible activity to CO is done. This is what we're doing now in Vietnam. This is the strongest bond. The next strongest bond would be where one takes the two CO apart a little, and then a little more in levels, to go a little into the side chains and then the next would the aliphatic bond where only 2 CO bonds are joined. I mentioned a while ago that I was very interested in plant systems and naturally, first of all the completely obvious, by which I mean plants that change colour when they are exposed to a specific influence. For instance, if you drop an apple it will have a brown patch the next day. And the colour has changed. Next slide please. It will not have escaped your notice that this is a banana, an Austrian one to be precise, because it is black and yellow. Thank you. So, this is an Austrian black-yellow banana, which has gone black because I have been maltreating it a little, because I dunked half of it in chloroform for a moment, and the next day it had gone Austrian. So what exactly did I do? As a young boy I – yes, it's very striking, isn't it? So, as a young boy I was very interested in this and discovered – what was partly, but not fully, known – what was actually going on here. Can I have the next please, that's the same again. There is a substance in the plant, some kind of a substance, the two hydroxyl groups, so that's the donor, a harmless affair, that promotes activity. And next to it is an enzyme, which takes two or one election from it, changing it all into a very strong oxidation agent. Now I would ask you to turn your attention to two great principles. One of the principles is what does nature do when it, it changes something, doesn’t it? Nature always kills two birds with one stone. It takes a system which exists in a living plant in a harmless form. Then, when something goes wrong, when the plant is compromised, the system and the enzyme attack the substance, accepting the two electrons and making an oxidisation agent, my electron acceptor. The second principle, which is very important and interesting, is that nature will defend itself if it suffers damage, that the damage not only creates damage but, at the same time, catalyses a system which rectifies the damage or protects us against it. You all know this from real life, if you go swimming, and if on that day the sun is shining brightly, then the sun's rays will damage your skin a little, which will then turn red, won't it? And then there is a system which also contains a phenol and an enzyme, which are both delicately kept apart, a separation which can be very easily destroyed. Sunshine is damaging to them and adversely affects the separation, the things come together. The culture oxidises. Phenol creates a pigment, and the pigment defends you against the sun. This is a fundamental principle of nature. Very, very ingenious. This is all around us, if you cut yourself and bleed, the cut activates a system, an enzyme system again, which creates fibrin to stop the blood which then ceases to flow. As I said before, you will frequently come across this system. A wonderful system. Our Creator, if we have one, must have had a lot of fun with it. Take the example of a plant that gets bacteria – it thinks now I will have an excellent Bavarian breakfast, and it begins to eat, at the same time activating the system that kills it. It has fallen into a trap. Nature has set a trap for the bacteria, which protects at the same time. So, this is the story of plants, known as polyphenol oxidase. The essence is naturally contained in the undamaged plant where the enzyme needs to be separate from the substrate and this separation must be very fine so that the most minute, the tiniest pathogen can be destroyed. Enzyme and substrate join forces and trigger the whole process. Once I had found out a little about what happens to these bananas, I ... half the plants are plants which changes colour. The other half doesn't change at all, such as lemons or oranges. You can chuck them on the floor, they won't go black. So, why not? This is something I worked on for many years without any success in solving the problem. My endeavours and my time were not entirely wasted as my work led me to the chance discovery that these systems contain a strong reducing agent. Naturally, as a good boy, I isolated this, finding what you know today as ascorbic acid. Ascorbic acid is a strong reductant which can always maintain phenols in a reduced state. Precisely, in recent times I have turned my attention to this system again and discovered that these plants have this kind of system. It is very similar to this. However, when two acids are set apart, the result is not black, solid or liquid – it remains colourless. There can be no bond with proteins. Consequently, this is simply what we have: these things are present in the plant and held in a reduced state by the ascorbic acid. If the plant is damaged, the ascorbic acid is oxidised away. It disappears and is no longer active. These substances then autooxidise to become dizinon, to paradizinon, and it kills the bacteria. So the same story again. It now becomes quite clear. The animal system, however, would not be able to function in this scenario. Plants are much more robust. The animal system is very unstable and extremely sensitive. It would not be able to work with this kind of conjugated double-bond system. These are frightfully strong acceptors which will only tolerate aliphatic dicarbonyl, two COs. The simplest aliphatic compound – next slide please – is the simplest dicarbonyl with a methyl, which is a glyoxal derivative. If one now wants to combine two COs, the question that needs to be asked is what type of CO do I want to attract, there are two different types. There is the ketone CO, which is simple CO, C here and C there, which has excellent thermodynamic properties. In chemical terms, however, it is very lazy and inert. The other type is where C is replaced by hydrogen, which is then an aldehyde. Aldehyde is in thermodynamic terms not as good. It is, however, highly reactive. The ideal would therefore be an aldo-ketone. Naturally, a ketone-aldehyde where one CO is like this and the other like that. And the simplest ketone-aldehyde is the one where a simple methyl group is attached, which would be methylglyoxal. This makes it all very exciting. So what's so hot about such a silly molecule? What is so exciting is that all living cells, all of them that we so far know of, are composed of an extremely active enzymatic system that converts methylglyoxal into lactic acid. Next slide please. Here I don't want to go into detail – there is methylglyoxal , and no - here is methylglyoxal which has two enzymes and a glutathione. All of them together react, and what we get in the end is lactic acid. The active ketone-aldehyde is gone, converted into tame lactic acid that can't do any damage. All living cells – thank you, that was sufficient – all living cells have extremely active methylglyoxal. And this is what is so wonderful, you see. In the first half of the century, our greatest biochemists, Neuberg and Dakin and Racker and Hopkins, were looking into this, and then slowly interest waned as no methylglyoxal was found. But there has to been something like methylglyoxal, as the enzyme, nature itself, it has no time for luxuries. An active enzyme is not just there for beauty's sake, there has to be something else There has to be a substrate, and this substrate must have an absolutely fundamental purpose. This substrate has to be a ketone-aldehyde, which makes it a good acceptor. This, in turn, has to do with a function connected with ketone-aldehyde and with the acceptor properties of ketone aldehyde. Yes, of course, given all the time I have spent studying plants it is natural that this was a growth inhibitor, right? The real issue at stake is this: All living tissue has a great propensity to multiply. The more active the system is, the more life there is, the more activity, and life wants to multiply, doesn't it? This is evident in the population explosion, life wants to multiply, multiply, multiply. As long as bacteria and lower-state things have nourishment, they will multiply rapidly. But this will not do in a multiple-cell organism. It all needs to be held in check in the interest of the whole. How is this living tissue restrained? Then the penny drops: perhaps this is a type of ketone-aldehyde. And perhaps it was difficult to find because not very much of it is needed. We have all ketone-aldehydes which can be tolerated, structured, chemical, and have found that in the minutest, one-thousandth molar concentration, all growth is kept in check, but they don't cause any harm. They are not at all poisonous in this concentration. They don't do a thing. Simply nothing, which means that nothing can multiply. Consequently, we arrive at a theory that our tissue is kept as it is precisely through ketone-aldehyde, which is present only in the minute quantity necessary and possible, which makes it impossible or very difficult to discover. Particularly, because ketone-aldehyde is highly reactive and binds easily with sulphur. As a result, ketone-aldehyde was only discovered in tissue in the very recent days. Now I want to go one step further by saying: Assuming that everything I have said is true and that in life, tissue is kept in equilibrium by a ketone-aldehyde, and assuming this is a highly active enzyme which serves to separate a frightfully delicate system from this kind of substrate and the wall separating the two was not properly structured, and the enzyme were to constantly eat away at the substrate? The cell would then start to multiply in a disordered fashion. And if a cell does this, it is called cancer, and this is what cancer is. At the present point in time, I believe that cancer is a state where enzyme and substrate are not precisely separated from one another, that the enzyme eats up the substrate in a senseless fashion, and the cell starts to multiply. Naturally, you then say: Well, if this is true, the cancerous cell must produce lactic acid because methylglyoxal always turns into lactic acid. And if we go one step further, we would say that it can only be the cancerous cell that produces aerobic lactic acid. This is precisely what Warburg discovered. Warburg found that it is only the cancer cell which produces lactic acid, aerobic. Naturally, his explanation of the production of lactic acid is different and I regret that he is not here, as he would be very angry with me. But because he isn't here, I can go on talking about it. I have looked at all of it, the literature, the proof that it comes from metabolism and not from methylglyoxal. It all proves nothing - it's too weak. I can't and don't want to continue talking about it without Warburg being present. So, let's go back to what I mentioned before about cancer. This is a theory, but nonetheless a good theory, a very good theory. A theory doesn't need to be true. There is only one condition: Please say whatever you want, but smile a little at the same time. So, I mention the theory with a little smile. It is a good theory because it is the first theory about cancer which is a bit physiological. It really is physiological and quite, quite simple. And secondly, there are many ideas about how to stop cancer, to prevent it from growing and make it go away. We are working on this very intensively now. I have this knowledge about cancer, which is in the process of evolving. I have revealed it because I wanted to show that these very abstract issues about electron clouds which we talk so much about, and that seem so pointless in biochemistry, may not only be deeply significant, but may also help us in solving humanity's most dreadful problems, at the beside of the those are ill. Thank you.

Albert von Szent-Györgyi on Curiosity Driven Discovery (in German)
(00:30:32 - 00:37:08)

The next Nobel Prize for vitamin research followed promptly in 1938, with the German organic chemist Richard Kuhn being distinguished for his work on the structure and synthesis of the vitamins B2 and B6. Kuhn later dedicated his 1952 Lindau lecture to the one foodstuff which was at the core of the discovery of the first vitamins: milk. Since most mammals live of nothing but milk during the first part of their life, a healthy mother’s milk must contain all macronutrients and micronutrients required for growth and development. This includes all vitamins, as Kuhn points out:

Richard Kuhn (1952) - The vitamins of milk (German presentation)

Normally, all mammals, for a short or longer length of time after birth, feed exclusively on their mother’s milk. Evidently, breast milk contains everything that is essential for the newborn to thrive. When, in 1909, Wilhelm Stepp in Strassburg and then Frederick Gowland Hopkins in England tried to raise young mice and young rats on a diet consisting of highly purified protein, fat, carbohydrate and mineral salts, they found it did not work. The animals died. By contrast, the animals developed well when milk was added to this diet. That is more or less what you might expect. But the experiments also led to an unexpected observation, namely that even “astonishingly small amounts of milk”, as Hopkins put it, were sufficient to supply the animals with what he himself called accessory nutrients, but which soon became known as vitamins. Milk can serve as an example to describe and explain nearly everything we have since learned about these vital substances: The distinction between fat-soluble vitamins, which separate into the cream during centrifugation, and water-soluble vitamins, which remain in the whey; methods of biological and chemical determination; enrichment and purification techniques, structural elucidation, and finally the synthesis of vitamins, their efficacy in treating deficiency disorders and the protection they offer against these and many others. Two vitamins in milk can be directly perceived, as they are pigments: Firstly, carotene, which separates out into the cream and is responsible for the yellow colour of butter; secondly, lactoflavin, which is water-soluble and gives whey its greenish-yellow colour. It is not my intention here to enumerate all the vitamins so far identified in milk individually or to discuss, with the help of long tables of figures, the differences in milk from various species, differences in vitamin content depending on diet, seasonal fluctuations or technical dairy problems. I wish to attempt, by drawing on just a few scientific findings arising from problems with milk, to show how they have gained relevance to other questions in chemistry and medicine. Two examples are particularly close to my heart, carotene, lactoflavin and a still uncharacterised factor found in human breast milk. Carotene was crystallised as early as 1831 by Wackenroder, not from butter but from carrots. However, it was not until 1928 that Hans von Euler in Stockholm showed it to be an essential raw substance, constituting the crucial factor in the fat-soluble phase in the pioneering experiments by Stepp and Hopkins. In an experiment with Edgar Lederer it was then discovered in 1931 that carotene can be separated into two clearly defined components. They are known as alpha and beta carotene. Together with Hans Brockmann, gamma carotene was later added. The method used for separating carotene into its constituent part is called chromatographic absorption analysis. And the extraordinary power of this method is reflected in the fact that it separated three isomeric carbohydrates, all of which have the same molecular formula: C40H56. The principle of chromatography was described by Schönbein in 1861 and in another form by the Russian botanist Zwet in 1906, but it had only been used occasionally as a qualitative tool for analytical observations. The separation of carotene into its constituents showed that the method is suitable for preparing organic compounds in their pure state. Since then, there has grown an almost bewildering array of applications for this tool. From my own institute, in particular Alfred Winterstein, Hans Brockmann and then Theodor Wieland made important methodological contributions. The technique has also been used on fluorescing compounds, on colourless compounds, on liquids and on gases. It has also found use in inorganic chemistry, for example, the separation of rare earth elements, a problem to which many outstanding chemists had devoted decades of their lives, which can now often be accomplished relatively easily by chromatography. And the same applies to the separation of uranium. And the principle of the method was specifically refined during World War II in England by Consden and Martin in the form of paper chromatographic analysis, which now appears to be irreplaceable for many problems in chemistry as well as biology and medicine. The question has occasionally been asked how such a simple and versatile method could be ignored for so long. Specifically, why Richard Willstädter, in his investigations of chlorophyll, did not use Zwet’s method, who had already described the separation of green pigment from leaf extracts into two components. Let me answer this question as follows. Willstädter was familiar with Zwet’s experiments, and he carried out the separation of chlorophyll into its A and B components according to Zwet’s description. And yet he rejected the method. The reason is that during the absorption of chlorophyll on silicic acid, on calcium carbonate, on aluminium oxide and on other adsorbants that Zwet had recommended, chlorophyll A and chlorophyll B undergo changes of a chemical nature that Zwet had not noticed, but which Willstädter did. During adsorption, the chlorophylls lost the property of turning brown after the addition of alkali. They lost what is known as the brown phase, and, for this reason, Willstädter believed that the method was unsuitable for isolating natural pigments. Among the numerous adsorbants Zwet recommended was, however, sucrose, on which, as Winterstein later showed, both chlorophylls remain intact. But sucrose, of all the substances, appears to have been ignored in the investigation by Willstädter’s laboratory. Of all the vitamins that occur in milk, probably only one was first actually isolated in its pure form from milk, namely lactoflavin. It occurs in nature, partly in a free state, dialyzable, and fluoresces yellowish-green, partly bound to proteins in an undialyzable and nonfluorescent form known as flavoproteins. Studies carried out jointly with Paul György and Theodor Wagner-Jauregg showed that lactoflavin has the same vitamin activity whether it is bound to protein or not, the amount of lactoflavin required to achieve one growth unit in young rats, that is, a weight gain of 40 grams in 30 days, is 10 grams lactoflavin. For lactoflavin later synthesised with Friedrich Weygand, the exact same activity was found. At the time, Otto Warburg had already showed that a yellow ferment occurs in yeast which acts within a system that oxidises glucose-6-phosphoric acid ester to gluconic acid-6-phosphoric acid ester, so that the activity of the ferment can be tracked by observing the oxygen consumption. By exposing his still crude ferment solution to light, Warburg obtained a chloroform-soluble fragment and the same fragment is obtained when the vitamin we isolated by crystallisation from milk is exposed to light. This observation led to the concept that vitamins are building blocks of ferments. Subsequently, this concept has been proved more precisely and in greater detail. The concept of a vitamin as a building block of a ferment explained for the first time why vitamins are active in such “astonishingly small amounts”. Because ferments, as catalysts, even in small quantities, are able to convert a large number of organic substrates in cells, and if a vitamin is a building block of such a ferment, it too, in small amounts, would result in high substance conversion rates. Theorell had found that Warburg’s yellow ferment could be split by the action of dilute acids into a colourless protein component and a phosphoric acid ester of lactoflavin. Together with Friedrich Weygand and Hermann Rudy, a complex multi-step process was used to obtain lactoflavin-5-phosphoric acid in a fully synthetic form, and it was possible to combine this synthetic lactoflavin-5-phosphoric acid ester with the protein component to obtain a fully catalytically active yellow ferment. Other flavins that do not occur in nature but can be synthesised have similarly been converted to synthetic yellow ferments by combining them with protein. The importance of the phosphoric acid residue for linking the vitamin to the specific protein body was illustrated in these experiments. The next picture shows you that the vitamin itself, the lactoflavin, has a certain affinity for the protein body. But this affinity is much smaller than that of lactoflavin-5-phosphoric acid ester. Here time is plotted, here oxygen consumption in cubic millimetres^2 in the enzymatic reaction. whereas 30 and 150 grams of lactoflavin bound only to protein, which dissociates much more strongly, is needed to even approach similar reaction speeds. The concept of a vitamin as the building block of a ferment was also recognised two years later by Karl Lohmann of the Meyerhof Institute in Heidelberg for vitamin B1, the antineuritic vitamin, in the form of chlorophosphoric acid ester, which forms a building block of carboxylase, an enzyme that plays a key role in splitting carbon dioxide from alpha-ketocarbonic acid. Vitamin B6 was also shown to have a similar relationship to ferments that split CO2 from amino acids. The generalisation of this concept became so widely accepted that as soon as a new vitamin was discovered, the question was asked in which ferment it acts as a building block. But this concept has not been proved in detail for the entire group of fat-soluble vitamins. It is questionable whether it will prove generally valid for this area. Although we know from George Wald’s elegant experiments that, for example, a cys-aldehyde of vitamin A forms visual purple in our retinas after combining with a specific protein body. But we also know that this function is definitely not the only one vitamin A has in the body. Lactoflavin is a vitamin not only in higher animals and for humans, it is also vital for many microorganisms, a growth factor, for example, of many lactic acid bacteria, whose activity is manifested when milk turns sour. This formation of lactic acid from carbohydrate also plays a key role in our muscles whenever work is performed. And the mechanism of lactic acid production was the object of a long series of admirable experiments by Otto Meyerhof. If I dedicate a few words to Meyerhof’s work here, I do so to convey the words this great physiologist said to me shortly before his death. I also do so to pave the way for an understanding of a specific lactic-acid-forming organism that we will now focus on. According to a long-standing principle formulated by Adolf von Baeyers, when oxygen combines with organic compounds, it tends to go where there is already oxygen. In the case of fructose with its 6 C atoms, we might therefore expect the C1 atom, which is at the highest oxidation state, namely the aldehydic state, to be the one to form CO2 during alcoholic fermentation or a carboxyl group of lactic acid during lactic-acid fermentation. But according to experiments by Otto Meyerhof, that is not the case. Consider the 6 C atoms of fructose here on the left, the first C atom of which is an aldehyde group, while each further C atom carries an alcoholic hydroxyl group. In a nutshell, Otto Meyerhof’s experiments showed that during lactic acid formation this first C atom is reduced to a methyl group of a lactic acid molecule and that the carboxyl group of both moles of lactic acid come from the third and fourth C atoms of fructose. The accuracy of Meyerhof’s concept can be easily proved today with the help of radioactive indicators. Mr. von Hevesy explained the principle to you this morning. The result was a resounding confirmation of Otto Meyerhof’s views. In this case it is possible to start with a non-radioactive pentose and to add the first C atom in radioactive form. In this way you obtain a fructose in which only the C1 atom is radioactive. On the other hand, if, for example, you grow sugar beets in an atmosphere of radioactive CO2, you obtain a sucrose from which you can extract fructose and glucose in which nearly all the radioactivity is located on the third and fourth C atoms. Thus, if we use glucose in which only the C1 atom is radioactive, we will find practically the entire activity in the one methyl group of lactic acid. In the case of alcoholic fermentation, the relationships are such that these two methyl groups from two molecules correspond to ethyl alcohol, and these two are released in the form of CO2. But last autumn a paper by Gonzales in Texas appeared which Mr. Meyerhof discussed with me in detail. The experiment was superbly conducted. It showed that another microorganism, Lactobacillus mesenteroides, breaks down fructose in such a way that 50% lactic-acid fermentation occurs, namely one mole of lactic acid is formed, as here, but instead of a second molecule of lactic acid, one mole of CO2 and one mole of ethyl alcohol are produced. And it was found that even when only the C1 atom in the fructose was radioactive, the entire radioactivity was found in the CO2. That is entirely at odds with Meyerhof’s scheme. It would have been understandable if the ethyl alcohol here had arisen from the first and second C atoms and the CO2 from the third. But that is definitely not the case. Meyerhof therefore concluded that his scheme is probably generally valid for so-called homofermentative lactic-acid producers, that is, for those microorganisms that form two moles of lactic acid from one mole of sugar. And that the fundamentally different mechanism used by heterofermentative microorganisms to break down carbohydrates, of which I’ve listed three, requires further research. Here, the breakdown scheme described has been clearly proved. For III and IV it has not been proved, an example of aldehyde, which produces, in addition to one mole of lactic acid, one mole of acetaldehyde and one CO2. The table shows another microorganism, Lactobacillus bifidus, which is also heterofermentative. It produces one mole of lactic acid and one mole of acetic acid. Lactobacillus bifidus was first isolated by Tissier in Paris in 1899 from the faeces of a breast-fed infant. Science is greatly indebted to the Heidelberg pediatrician Moro for his research. It is a highly sensitive microorganism. Its name is meant to express its shape, as it is branched, or bifurcated, at the ends, hence bifidus. It is Gram-positive, taking on Gram stain readily. The faeces of a breast-fed infant contains almost only Lactobacillus bifidus. When an infant is fed cow’s milk, the quantity of Lactobacillus bifidus rapidly declines; when human milk is given again, their number rebounds. It is an anaerobe, meaning that it has a poor tolerance of oxygen and this has created numerous problems with regard to its isolation. According to Hotchkiss, it stains bright red. This staining is achieved by first exposing the microorganism to a solution of sodium periodate, which splits carbohydrates into aldehyde groups. In the second step it is exposed to fuchsin-sulfuric acid, which makes the aldehyde groups visible by staining them red. Under the microscope, Lactobacillus bifidus cultures look like alphabet soup with nothing but capital Y's floating in it. But this structure can be altered, as I mentioned, quite easily, not only under the influence of oxygen, and the changes are not only expressed morphologically. Rather, it has been shown that along with many other changes, a characteristic physiological property can also change suddenly. Instead of optically active lactic acid, only racemic lactic acid is produced. Just three years ago researchers in the United States succeeded in establishing a clone of Lactobacillus bifidus for growing cultures, in which all the cells are derived from one and the same parent cell. Subsequently, Paul György isolated a mutant Lactobacillus bifidus strain from the faeces of a breast-fed infant. This strain had the property of growing only if human milk was added to the nutrient medium or solution. When cow’s milk was added, the mutant strain did not develop. This mutant has long been referred to by its laboratory name, 212A. It was later registered under the name Lactobacillus bifidus var. Penn. Penn is short for the University of Pennsylvania in Philadelphia, where the strain was first isolated. The next picture shows some known differences between the composition of human milk and cow’s milk. Most notably, human milk contains only half as much protein as cow’s milk, so that to prepare infant formulas, cow’s milk is often diluted by around 50% so that it contains the same protein content. And then sugar is added to bring it up to 6.6%. And in dozens of variants, you can also add other vitamins, mineral salts and so forth. For Lactobacillus bifidus there appears to be some hitherto unknown essential difference in the chemical composition of human milk and cow’s milk. And although it was not initially clear what impact the nutrient requirement of such a mutant has on an infant’s overall intestinal flora, I believe that Paul György’s discovery has enriched and invigorated an important chapter in pediatrics by raising new questions. In any case, it led to a test, by means of which it became possible to chemically track down an active substance in human milk. And the test is carried out as follows: The gas atmosphere is a mixture containing 90% nitrogen and 10% CO2. These bifidus strains are CO2-dependent. In addition, some hydrogen is added to the atmosphere and then a glowing platinum mesh burns off the remaining oxygen, so that growth takes place under strictly anaerobic conditions. The next picture shows the first half of the synthetic nutrient solution that György developed together with Dr Rose. A litre of this semisynthetic nutrient solution contains Also a number of amino acids, which are listed here. But the nutrient medium has so far never been fully synthesised. You still have to add a casein hydrolysate, and not an insignificant amount of it, in order to carry out the following determinations. The next picture shows more mineral salts and vitamins required for this test: potassium phosphate in relatively large quantity, iron, manganese and the vitamins shown here. All of this is sterilised, and only then is one gram of ascorbic acid added to one litre. Now everything is ready for the test. To determine the activity, the total acid, that is the sum of lactic acid and acetic acid produced by the growing bifidus cells, is potentiometrically titrated. Here you see a comparison of acid production in 40 hours at 37° after the addition of human milk and cow’s milk. Here it states how many tenths of a cubic centimetre of normal acid are produced. This word could also have been written with two g’s. You can see that in the case of human milk just 0.023 cubic centimetres is enough to produce 2.2 cubic centimetres of acid, whereas with cow’s milk one cubic centimetre, fifty times more, is needed to produce the same quantity of acid. Based on these figures, we would say that cow’s milk has only a fiftieth of the activity of human milk. In total, under the conditions used, approximately 18 tenths of a cubic centimetre of normal acid is produced from the lactose present, so that half that quantity, half the maximum possible lactic acid, or 9 tenths of a cubic centimetre, has been defined as one unit of activity. In other words, one bifidus unit is the quantity of bifidus-active substance needed to produce 9 tenths of a cubic centimetre of normal acid. The next picture compares the activity of milk from various animal species. You can see the activity is very low in all strict herbivores. One unit for such and such cubic centimetres of milk, the smaller the number, the greater the activity, and the last column shows the relative activities, taking 0.06 cubic centimetres for average human milk as equivalent to 100. You can see that guinea pig milk is practically inactive, cow’s milk, sheep’s milk and goat’s milk have very little activity, just 2 to 3% the activity of human milk. Pig’s milk is somewhat more active, and the most active of all the animals listed here is rat’s milk and rat’s colostrum, which is the very first milk the animal secretes after giving birth, and is not only more active, but around twice as active as average human milk. The most active we know is human colostrum, the first milk of a woman. There is also a value for cow’s colostrum. The case is as follows. At the time of birth, cow’s milk also has considerable activity. But it falls from this value of 40 to around 2 to 3% in the space of just two to three days. The activity of human colostrum declines much more slowly to reach, over the course of many, many weeks and months, a relatively constant level of 0.06 cubic centimetres of human milk per unit. We have also studied women who have breast-fed for a whole year or longer. And we found that during such long lactation, the activity continues to decline to 0.08, 0.12 cubic centimetres. But what I also want to mention is that precisely in those women who have breast-fed for so long, a bifidus activity of 0.02 cubic centimetres per unit was still found six to ten weeks postpartum, a value that is otherwise found only in human colostrum. Besides milk and colostrum, human sperm is also highly bifidus-active. Sperm from a bull or bulls’ testicles is completely inactive. The next picture shows examples of the distribution of the bifidus-active substance in the human body. At the top is again human colostrum at 0.01 to 0.2 cubic centimetres, with a mean of 0.015. Then milk with 0.02 to 0.15, or 0.06 on average. Meconium, the intestinal content of a newborn infant, in a 10% suspension is also highly active. Sperm 0.07 cubic centimetres on average. Gastric juice, saliva, urine, I’ll have something special to say about them later. Tears too. If you want to compare this figure with those for milk, you have to take into account the fact that milk contains approximately 12% dry substance, compared to only 0.5% organic substance in human tear fluid on average. Calculated on the basis of dry substance … [stops here]

Richard Kuhn on the Role of Milk in Vitamin Research (in German)
(00:00:12 - 00:02:58)

Another five years later, Henrik Dam and Edward Doisy shared the 1943 Prize in Physiology or Medicine, being rewarded for the discovery and structural elucidation of vitamin K.

The Special Case of Vitamin B12
Vitamin B12 takes a special role amongst the vitamins. It is the only vitamin that contains a metal atom (cobalt), as part of its structure. With a weight of 1580 Dalton it is the heaviest of the vitamins. It is the last vitamin to be structurally characterized. And it was linked to a Nobel Prize before it was even known to exist.
This Prize was given in 1934 to George Whipple, George Minot and William Murphy “for their discoveries concerning liver therapy in cases of anaemia". The scientists had shown, that certain fatal types of anaemia, a disease caused by the lack of red blood cells, could be treated by having patients eat liver. Thousands of lives could be saved by this method. However, when the Prize was awarded, the reasons for this success were unclear and only in 1948 the actual active agent, vitamin B12, could be isolated in crystalline form.

It then took another seven years before the British biochemist Dorothy Hodgkin was able to determine the correct structure of the crystalline compound by using the emerging technique of
x-ray diffraction crystallography
. Hodgkin was awarded the 1964 Chemistry Nobel Prize, inter alia for this achievement. It was the last Nobel Prize linked to the discovery, structural elucidation or synthesis of a vitamin so far.

Vitamins: A Story with an Open End
But of course, the story of vitamins does not end here. In the years following their discovery, researchers began to work towards an understanding of the biochemical role of the individual compounds. It was found, for example, that vitamin A is contained it the retina and directly involved in the visual process. This insight led to the 1967 Nobel Prize in Physiology or Medicine, which was given to Ragnar Granit, Haldan Hartline and George Wald "for their discoveries concerning the primary physiological and chemical visual processes in the eye".

Besides such fundamental work, numerous studies were concerned with determining the potential of vitamins in preventing or curing mental and physical diseases. Unfortunately, not all of these studies were conclusive and by no means is our knowledge on the biochemical functions or health effects of vitamins complete. It is thus not surprising that the wellknown recommended daily allowance (RDA) or dietary reference intake (DRI) are, in part, subject to heavy discussion.

Linus Pauling and Vitamin C
One of the most prominent critics of these RDA values was double Nobel Laureate Linus Pauling. Pauling claimed that vitamin C megadoses (around 10 gram per day, or 12500 % of the current EU-RDA) would be suited to treat cancer and support health in general. He himself took at least 10 gram of vitamin C per day for more than 20 years. In his 1981 Lindau lecture, Pauling explained how he conceived his 10 g/day recommendation:

Linus Pauling (1981) - Vitamin C in the Prevention and Treatment of Cancer

I am going to speak about vitamin C and cancer. I hadn't planned to work on cancer, work that I began, in fact, 10 years ago, nor to work on vitamin C or other vitamins as I began around 16 years ago. Instead I got into these fields by accident, or through some concatenation of circumstances that just left me, led me into these fields. I started to work on haemoglobin after having worked on simpler molecules for 14 years in 1936, and then the next year I began some work, together with my students of course, in the field of immunology. Then in 1945, I had an idea that sickle-cell anaemia might be a disease of a molecule, rather than a disease of a cell. Dr. Harvey Itano, a young physician, came to study with me and he and I began to check up on this idea. In 1949, together with two other students, Singer and Wells, we published a paper; "Sickle-Cell Anaemia, a Molecular Disease". After being with me 8 years, Dr. Itano was ordered as an officer of the public health service to move to Bethesda. And I decided that I should give up work on the hereditary haemolytic anaemias. It didn't seem to me very sensible of me to be competing with such an able and vigorous young investigator as Dr. Itano. So I thought, why shouldn't I look at other diseases to see whether or not they are molecular diseases? And they might as well be important diseases, because nobody else was working in the field, except by that time there were a good number of haematologists studying the haemoglobin anaemias. I thought, well, I might work on cancer. Or I might work on mental disease. And I rejected cancer for two reasons; one, it seemed to me that it was just too complicated a field for me to be involved in. And second, many people, many investigators were carrying on studies in the field of cancer, whereas in 1954 very few people were working on mental disease. It was of mental disease, our study is on schizophrenia and mental retardation that got me into vitamins. After about 10 years of work in this field I ran across some papers, publications, by doctors Hoffer and Osmond in Canada, Saskatoon, Saskatchewan, Canada. They made a report that really astonished me. They said that they were giving large doses of nicotinic acid or nicotinamide to schizophrenic patients. I knew, of course, that a little bit of nicotinic acid or nicotinamide must be ingested day after day, 5 milligrams, perhaps a little pinch, to keep a person from dying of pellagra. So I knew that these substances, the pellagra-preventing factor, are very powerful substances. And yet these substances are so lacking in toxicity that Hoffer and Osmond were giving a thousand or ten thousand times this physiologically effective amount, or pharmacologically effective therapeutic amount, to schizophrenic patients. Nobody knows just how toxic they are, people have taken 100 grams a day, or even more, without any serious side effects. I thought, how astonishing that there are substances of this sort that have physiological activity over a tremendous range of concentration, 1,000 fold or 10,000 fold range of concentration. In fact I found that Milner had been giving large doses of vitamin C in a double-blind experiment to schizophrenic patients and he reported that several grams, perhaps the thousand times the amount that will prevent scurvy in most people, several grams of ascorbic acid is also effective, more effective than a placebo, for these schizophrenic patients. The idea, that such substances exist, which are effective in one way or another over a tremendous range of concentrations, caused me to decide that this field of medicine deserved a name. I invented the word 'orthomolecular' to describe it. The right molecules are the molecules that are normally present in the human body, and the right amounts are they amounts that put people in the best of health. Well, I was interested in infectious diseases in relation to vitamin C originally, but in 1971 Charles Huggins asked me to speak at the dedication of his new cancer research laboratory. I thought, I must say something about cancer. So I remembered that I had read a book published in 1966 by Ewan Cameron, a surgeon in a hospital in Scotland who had been a general surgeon who had however all of his life as a surgeon been interested in cancer. He formulated a general argument in this book, "Hyaluronidase and Cancer". His argument was this - he said "the body has protective mechanisms, the immune system for example. If we could potentiate these, the natural protective mechanisms, they might provide additional protection against cancer". As Professor de Duve pointed out, we have, in many patients, most patients with cancer, circulating cells, but not all of them develop metastases. Those, whose immune systems are functioning well, have a smaller chance of developing metastatic cancer than those whose immune systems are not functioning well. Well, I thought, we know one thing about vitamin C, it is required for the synthesis of collagen. If then, persons with cancer were to be given larger amounts of vitamin C, they would be stimulated to produce more collagen fibrils in the intercellular cement that holds the cells in normal tissues together. These tissues might become strengthened in this way to such an extent that, as Cameron pointed out, without mention of vitamin C, to such an extent that they could resist infiltration by the growing malignant tumour. Dr. Cameron saw a newspaper account of my talk and wrote asking how much vitamin C to give. I replied he should give the patients 10 grams a day, 10,000 milligrams, 200 times the usually recommended amount. He began, cautiously, with one patient in Vale of Leven Hospital, Loch Lomond side, Scotland, and was astonished by the response of that patient, to such an extent that he gave 10 grams of vitamin C a day to a second terminal cancer patient, an untreatable patient receiving no treatment other than the vitamin C and narcotics to control pain. And then a third and fourth and more and more patients as he became more and more convinced of the value of this substance for patients with cancer. I, me, have started out now with the first slide. Well, I think, and perhaps this is the message that I should emphasise, I think that the people in the field of nutrition, the scientists in the field of nutrition, which up to 15 years ago seemed to me to be a terribly boring subject, have been off on the wrong track. They have said that a vitamin is needed, it's an organic compound needed in small amount to prevent death by a corresponding deficiency disease. And they have striven, very vigorously, over a period of 40 or 50 years, to find out just how much each of these substances is needed to keep people from dying. I believe that the problem that should be attacked is that of finding the intake that would put people in the best of health, not just the amount that will keep them from dying. The nutritionists refer to their recommend dietary allowances, RDA, by saying that these are the amounts that will prevent most people from developing the corresponding deficiency disease. Most people in ordinary good health. What they should say is that, it will prevent most people who are in what is ordinary poor health from dying from the corresponding deficiency disease. To be in what ought to be ordinary good health, they need to be ingesting the optimum amounts, the proper amounts, of these valuable substances. Next slide. There's an interesting difference between vitamin C and the other vitamins. The other vitamins, thiamine, pyridoxine, riboflavin, vitamin A and so on, are required by essentially all animal species. Vitamin C is not required by most animal species, 99% or more of animal species synthesize ascorbate, they do not rely on the dietary sources of the substance. If I ask, why do these animals continue to synthesize vitamin C even though they may be getting large amounts by ordinary standards in their diet, several grams a day for an animal the size of a man, the answer surely is that they continue to synthesize ascorbate because the amounts they get in their diet are not enough to put them in the best of health, not enough to put them in the fittest condition in the environments in which we live. Next slide. Man is one of the few unfortunate species of animals who are in rather poor health generally because of not having as much ascorbate as corresponds to the best of health. When I looked at 150 raw natural plant foods, taking the amounts that would give 2,500 kilocalories of energy, I found that for thiamine and other vitamins there was perhaps 3 times or 5 times as much of the vitamin as is now recommended, as you get in modern diet on the average, but 50 times as much vitamin C as is recommended. And I thought, this is an indication that larger amounts of vitamin C are needed because animals are getting these larger amounts but continue to make ascorbate. Next slide. The next slide please. Another interesting fact is that the committee that recommends the diet, the food for monkeys, experimental monkeys, recommends 70 times as much vitamin C. Monkeys also require exogenous vitamin C, they are primates and all of the primates require this vitamin. I think that this is understandable, monkeys are very valuable, experimental monkeys, if you've spent months carrying out studies with them and then suddenly your monkeys die, it's a real tragedy, so that a great effort has been made to find out how much vitamin C will put the monkeys in the best of health. No one has gone to the effort to carry out corresponding studies for human beings. Next slide. Well, I mentioned that these most species of animals synthesize ascorbate. The amount they synthesize depends on the size of the animal; small animals produce a small amount, large ones a large amount. Proportional to body weight, not to surface area, 2/3s however of the body weight, but to body weight. And the amount produced by different animal species is between 40 and 400 times the usual recommended intake for human beings. Averages 10 grams per day, per 70 kilogram body weight, that's why I wrote to Ewan Cameron, to say that 10 grams a day is the amount that he should try. I might say that the pharmacologists sometimes say that 50 milligrams a day of vitamin C per day is a physiological intake and that 10 grams a day is a pharmacological intake, that the vitamin is being used as a drug. I would say that 10 grams a day is the proper physiological intake and 100 grams a day might be called the pharmacological intake. And people have taken that amount, people have received 125, 150 grams of sodium ascorbate a day by intravenous infusion to control serious diseases, without any side effects, and have taken similar amounts by mouth. Next slide. So, this is the conclusion that I have reached. I have already stated it. Next slide please. Well, vitamin C is required for synthesizing collagen and I think it might well strengthen the normal tissues. Next slide. The reason that it is required is that collagen is formed from procollagen by hydroxylation of prolyl and seryl residues and there are other hydroxylation reactions, this one and other similar reactions do not take place except with use of vitamin C. Next slide. Well, collagen, the value of increased intake of vitamin C, under several circumstances, has been known for a long time, and surgeons for over 40 years have been recommended in the better surgical text books to give all surgical patients 1 gram or 2 or 3 grams of vitamin C per day in order to facilitate wound healing, healing of broken bones, of burns, to take care of peptic ulcers, periodontal disease, physicians have known, and dentists too, have known about this, they don't all practice it, but it's been known. Next slide. Here is a reference to Ewan Cameron, my associate for 10 years now in this work, and to his book "Hyaluronidase and Cancer". Next slide. And I argued then, in 1971, stated on this slide, that the increased synthesis of collagen might strengthen normal tissues to a significant extent. Next slide. Since then, a large amount of information has been gathered about the relation between intake of vitamin C and various aspects of the immune protective mechanisms. Vallance, Fagan, Yonemoto, others have shown that antibodies, IgG and IgM are produced in larger amounts with the increased intake of ascorbate. Fagan showed that a component of complement involving collagen-like sequences of amino acids, as shown by Prof. Porter, is produced in larger amounts, the blastogenesis of lymphocytes occurs at a greater rate, the activation of cytotoxic macrophages has been shown to be increased and interferon production is reported to be greater with a greater intake of vitamin C. I might mention that there's been a tremendous amount of interest in interferon for the treatment of cancer. One important point here is that to treat a patient with interferon costs about a thousand times as much as to treat him with ascorbic acid. Dr. Cameron says to people who ask about interferon, take ascorbic acid and synthesize your own interferon. Next slide please. This is the only study on vitamin C that has been carried out in the National Cancer Institute of the United States, Yonemoto, Chretien and Fehniger gave vitamin C, 5 grams a day for three days, to volunteers. The rate of blastogenesis of lymphocytes under antigenic stimulation doubled with this intake. When 10 grams a day for three days was given the rate tripled, and when 18 grams a day for three days was given the rate quadrupled. It's known that a high rate of blastogenesis of lymphocytes in the cancer patient is correlated with a better prognosis, longer survival, than a lower rate of blastogenesis. Next slide. Ascorbate seems to have a significant prophylactic value. Here I have listed seven studies relating to vitamin C and cancer, studies in which when a number of environmental or nutritional factors were correlated with the morbidity from cancer, vitamin C turned out to have the highest correlation coefficient, negative of course, to be the factor that seemed to be most strongly related to morbidity from cancer. Next slide. An interesting study was carried out by Dr. DeCosse and his associates. DeCosse is now the head of surgery in the Memorial Sloan-Kettering Cancer Centre in New York City. He found that 3 grams a day of vitamin C given to patients with familial polyposis caused the polyps to disappear in half of the patients. I've suggested that he give 10 grams a day in a new trial, which is underway, but he is sticking with 3 grams a day because of his worry about toxic side effects of large doses. Well, there just aren't any toxic side effects of large doses of vitamin C, talk about kidney stones has essentially no basis whatever, no cases in the medical literature. Damage to the liver doesn't occur, although it's sometimes mentioned without references. Next slide please. Bruce in Toronto has used the Ames-method of testing for mutagens to study faecal material, the contents of the lower intestinal tract and there are many mutagens that show up, and they, of course, when tested by, with much difficulty, various mutagens for carcinogenic activity have usually been found, When vitamin C is given by mouth to patients, the number of mutagens in the faecal material is much less. This is presumably a mechanism for preventing cancer of the lower gastrointestinal tract. I find, when I take 10 grams a day, which is the amount that I do take, that half of the vitamin C, Presumably then providing protection against this tract and in particular, of course, preventing the formation of nitrosamines, which are a cause of gastric cancer and other cancers, but also destroying other mutagens in the materials in the gastrointestinal tract. Of this 30%, 1.5 grams is eliminated in the urine, and provides protection of the urinary tract on the way out, and the other 3 1/2 grams works throughout the human body. Next slide. Here are some clinical tests, all have been carried out that have been reported as yet, I made a mistake when I wrote the copy for this slide, Cameron's last study involved 300 terminal cancer patients treated with ascorbate, compared with 2,000 matched controls in the same hospital. With these studies, a hundred against a thousand matched controls, there was essential random distribution of patients between Dr. Cameron on the one hand and the other surgeons and physicians in the same hospital on the other. Over a period of time when Cameron was giving the patients with terminal cancer, untreatable cancer, vitamin C, and the other surgeons and the physicians were not, so that we had a sort of randomized allocation of students of patients to the two groups. Morishita and Morata are associates of our institute in California, Morata has worked there two summers and their work is carried out in a hospital in Japan. Next slide. The first thing that Dr. Cameron and his collaborators, the surgeons working with him, noticed was that the patients feel better when they receive ascorbate. Cancer patients usually are pretty miserable, don't feel well, they have poor appetites and don't eat well. These patients lost their cachexia, they began to feel lively, feel well, have good appetites and then there were other responses that were noted. And later on, of course, it was found, next slide, it was found that they survived longer than the controls. This slide shows survival times of the hundred patients with untreatable cancer who received ascorbate, and the thousand matched controls. Ten matched to each of the ascorbate-treated patients who had also reached the untreatable stage when no therapy was administered to them except morphine or diamorphine to control pain. After the date of untreatability, the controls lived on the average 54 days, and the ascorbate treated patients lived on the average about a year, much longer. Of the controls only three in a thousand, 3/10 of 1%, survived over 400 days, 4/10 of 1% over a year after untreatability, whereas around 16% of the ascorbate-treated patients, who continued to survive. And these patients continued to survive for a long time, as much now as 8 years after having been considered to be terminal with an expected survival time of only a couple of months. Next slide. These are results of similar observations made in Fukuoka Torikai Hospital in Japan, Fukuoka Japan. Again, there's a low ascorbate group, receiving less than 5 grams of vitamin C per day, in Cameron's patients in Scotland the low ascorbate group received very little, perhaps 50 milligrams a day, and the high ascorbate group receiving more than 5 grams a day, an average of about 15 grams a day. The curves, the survival curves are essentially the same as for the study in Scotland. These graphs represent a breakdown of that comparison of a hundred, the first hundred ascorbate-treated patients in Scotland, and a thousand matched controls. Here we have seventeen patients with cancer of the colon who had reached the untreatable stage, compared with a 170 matched controls. The controls died off pretty rapidly, the ascorbate-treated patients lived on, a number of them here, With cancer of the stomach, bronchus and breast, the situation is rather similar. Next slide. Cancer of the kidney, rectum, bladder, ovary, there doesn't seem to be much difference in the response of patients with different kinds of primary cancer to ascorbate. There are some statistically significant differences, but it's a difference between living a year longer on the average and living 7 months longer. In general, I would say these patients, who have reached the untreatable stage and in Scotland, the untreatable stage for adult patients with solid tumours, gastrointestinal tumours and so on, do not receive chemotherapy so that these patients in general had not been treated with chemotherapy. In general, I would say that the evidence indicates that to give them ascorbic acid leads to greater survival time as well as better well-being during the period of survival, than treatment with chemotherapy does, the standard sorts of chemotherapy that are used now. Next slide. Another trial carried out was by Creagan, Mertel and others in the Mayo Clinic. The difference between that trial and the trials in Scotland and Japan is that 88% of the patients, We argued, Dr. Cameron and I, before the Mayo clinic trial was begun, that they should not use patients who had had their immune systems badly damaged by courses of chemotherapy, because of our feeling that vitamin C works largely by stimulating production of, by stimulating the immune system. Next slide. Well, our conclusions, with the possible, and I just quote from our book, the last sentences in our book concern vitamin C, in the management of all cancer patients from as early in the illness as possible. Next slide. We believe that this simple method, measure would improve the overall results of cancer treatment quite dramatically, not only by making the patients more resistant to their illness, but also by protecting them against some of the serious and occasionally fatal complications of the cancer treatment itself. We are quite convinced that in the not too distant future, supplemental ascorbate will have an established place in all cancer treatment regimes". Next slide. Now the advantages, it is an orthomolecular substance, every human being has vitamin C in his body, so long as he continues to live, has very low toxicity, no serious side effects, makes a patient feel much better, very low cost, it's compatible with most or all other methods of treatment, the exception being chemotherapy. Of course, I think that chemotherapy should be used with childhood cancer, leukaemia, Hodgkin's disease, possibly together with vitamin C, but my own feeling is that vitamin C for adults with solid tumours is probably preferable to chemotherapy. Well, I can end up by saying that in the United States, the medical profession, as an organized group, has not accepted these ideas. Individual physicians, I would judge have, because we get hundreds of letters and telephone calls from individual physicians who have developed cancer asking for more information. Thank you. Applause.

Linus Pauling on His Justification of Vitamin C Megadoses
(00:09:45 - 00:15:18)

To date (2013), it has not been proven that vitamin C megadoses are suited as a cancer therapy and the studies Pauling describes in his talk have been shown to contain systematic flaws. The Linus Pauling Institute at the Oregon State University, founded in 1973 by Pauling and colleagues, now distances itself from the claim that vitamin C is effective in cancer therapy and recommends a rather low daily intake of 400 milligram (or 500 % of the current EU-RDA) based on the “currently available epidemiological, biochemical, and clinical evidence” [4].

Signalling: Neurotransmitters, Hormones and the Sense of Smell
Leaving behind nutrition and metabolism, we arrive at another important biochemical domain of small molecules: the transduction of signals. Just as metabolic processes, chemical signalling is a pillar of life. It perpetually occurs on almost all scales – inside individual cells or in-between different cells of the same organism, between an individual organism and the environment, but also between organisms, be they of the same or different species.

Hundreds of messenger molecules, belonging to all kinds of compound classes are known to act in our bodies. However, whenever a signal has to be transmitted very rapidly or over a very long distance, the odds are that a small molecule is involved. This is due to several reasons: small molecules can be made and processed much faster than proteins, they can, in part, migrate through cell walls and, in the case of smells or pheromones, be distributed via air.

Neurotransmission
A classic example of rapid chemical signalling is neurotransmission. Today we know, that signal transduction between neurons is achieved by small molecule neurotransmitters such as glutamate, GABA, glycine, dopamine or serotonin. However, for a long time the crucial role of these small molecules was not realized and neurotransmission was assumed to be merely electrical in nature. Only in 1920, the pharmacologist Otto Loewi, then at the University of Graz in Austria, showed with a famous experiment that a chemical substance must be involved. Loewi himself described this experiment as follows [6]:

“The hearts of two frogs were isolated, the first with its nerves, the second without. Both hearts were attached to Straub canulas filled with a little Ringer solution. The vagus nerve of the first heart was stimulated for a few minutes. Then the Ringer solution that had been in the first heart during the stimulation of the vagus was transferred to the second heart. It slowed and its beats diminished just as if its vagus had been stimulated. Similarly, when the accelerator nerve was stimulated and the Ringer from this period transferred, the second heart speeded up and its beats increased. These results unequivocally proved that the nerves do not influence the heart directly but liberate from their terminals specific chemical substances which, in their turn, cause the well-known modifications of the function of the heart characteristic of the stimulation of its nerves.”

Together with the Brit Henry Dale, Loewi then went on to discover the first neurotransmitter, acetylcholine. Both scientists shared the 1936 Nobel Prize in Physiology or Medicine. Loewi also showed that after neurotransmission acetylcholine is broken down to two other small molecules, acetate and choline, by an enzyme called acetylcholine esterase. This is one of the fastest enzymatic reactions known. A single molecule of acetylcholine esterase, which is considered to be an “evolutionary perfect” enzyme [7], can hydrolyse around 16.000 molecules of acetylcholine per second [8]. Proteins cannot be made or degraded at similar rates.

Loewi was a pioneer of neuroscience and many others followed in his footsteps. At the 2011 Lindau Nobel Laureate Meeting, 1991 Nobel Laureate Erwin Neher, who had received a shared Prize for his work on ion channels, summarized the history and current knowledge on neurotransmission:

Erwin Neher (2011) - Signals and Signalling Mechanisms in the Central Nervous System

I think it is a challenge to keep up after these fantastic lectures this morning but I'll try anyway. My topic is signals and signalling mechanisms in the central nervous system and since I understand that this is a pretty diverse audience let me try to give a short introduction into the historical background regarding our knowledge on what happens in our brain. And of course this knowledge about bio-electricity, about signalling the brain starts with Luigi Galvani and Alessandro Volta in Italy. Luigi Galvani who showed that there is electricity in our bodies that frog muscles can be made to twitch when the nerve is stimulated by electric shock. About one hundred years later we got a very good impression on the structure of our brain when Ramon y Cajal showed that our brain is made up of these very delicate structures which we call neurons. And today we know that our brain is a network of about 1012 such neurons which are extensively connected with each other. Each nerve cell receiving input from up to about ten thousand neurons. Ramon y Cajal also developed ideas on the signal flow in the nervous system. When you look at his beautiful drawings you discover these little arrows everywhere. They show what his ideas about the signal flow was and one must say he had very good intuition. He was mostly right. Of course he didn't know what these signals were. But about at the same time Julius Bernstein showed what he called the "negative Schwankung" an electrical signal associated with nerve activity, which he was able to measure with this very difficult apparatus. And he also formulated his membrane theory which said the neurons are surrounded by a membrane and it's actually the voltage across this membrane which is responsible for signal and which changes in the way of this "negative Schwankung" when there is nerve activity. Now, 1952 Hodgkin and Huxley provided our understanding of the action potential of the nerve impulse by showing that actually when you depolarise a nerve something happens. When you cross a threshold the membrane suddenly becomes permanent with sodium ions. So even the electrochemical radiance across biological membranes and sodium rushes in and makes the inside more positive. But this is only a very short duration, soon after the membrane becomes permeable to potassium, potassium ions leaves the cell and this makes it again negative. Okay, so this more or less formed our understanding of what the brain is about. Each cell receives all its inputs mainly on its dendrites and adds up integrates its signals and once the threshold in a given cell has been surpassed the action potential is generated which then travels along the axon and signals to other cells. Now, the other part of course of the picture is what happens when such an electrical signal arrives, that's a nerve ending and signals to the receiving cell. This phenomenon synaptic transmission was first studied in detail at the neuromuscular junction. A special type of synapse when the nerve interacts with the muscle and gives its signal for the muscle to contract. There is a typical synapse at this neuromuscular junction. This is where Sir Bernhard Katz and colleagues studied this process of synaptic transmission in the 50s and 60s. What you see here is one of the basic discoveries which they made when they studied the signal in the muscle which is elicited by a nerve action potential. This is shown down here what happens is when the nerve impulse arrives there is a deflection. A positive signal which is the so-called synaptic signal generated by synaptic current, which then elicits an action potential a nerve impulse very much like the nerve impulse. Now what Bernhard Kutz did he looked at this at the baseline, he turned up the gain of his amplifier and observed something very strange namely that when you look at this baseline in high resolution you see all these little blips. So many researchers might dismiss this signal because it may be just some interference. But he decided to study it and what he found was when he reduced extra-cellular calcium concentration this nerve evoked signal became smaller and smaller and eventually at very low calcium concentration there was basically nothing left and the nerve impulse either did not illicit any signal or else elicited a signal of this kind which by itself happens spontaneously now and then. And this information of course when it was combined with the electro-microscopic evidence at the time that in the nerve terminals there are these little structures, these little granules called synaptic vesicles. This combined led to our current understanding of what synaptic transmission is, namely that a nerve impulse causes the influx of calcium ions. This was an underlying observation was that the strength of the signal is so critically dependent on extra calcium concentration. So this is calcium entering the nerve terminal as a consequence of the action potential, the calcium makes vesicles fuse with the plasma membrane and the vesicles are filled with neurotransmitter which diffuses to the postsynaptic membrane. The neurotransmitter opens channels in the postsynaptic membrane and so this again causes an electrical signal in the postsynaptic membrane. So we have this relatively complex process and electrical signal being translated into a chemical signal and back again to an electrical signal. Okay, so there is one very special feature of this kind of signal transmission which is very different in the brain as compared to a computer. In the computer the signal elicited by one transistor in the receiving circuit is always the same. But in neuroscience you observe basically everywhere you look that the synaptic strength which is the signal produced by a pre-synaptic action potential in the postsynaptic cell. So the synaptic strength changes in a youth dependent member. And neuroscientists are convinced that it is this plasticity, the constant reorganisation of the neuronal network which is at the basis of many of the very complex information processing tasks in the central nervous system. This plasticity occurs on many times scales, there are phenomena of long-term plasticities, so-called long-term depression and I think there is general agreement that this is the basis of learning and memory. But there is also short-term plasticity on the timescale of milliseconds to seconds which mediates basic signalling tasks like filtering adaptation and other things like gain control. So when we study the nervous system today we can principally take two approaches. Either the top-down approach starting from the higher functions and try to understand by lesion, by imaging how these functions are being generated by the element of the brain. Or else we can take the bottom-up approach which starts at the building blocks of molecules and tries to understand phenomena like nerve impulse synaptic transmission and so on. So my laboratory and my interest have guided me on the bottom up pathway. Already during my thesis - this is an illustration taken from my own thesis - recording from a giant neuron of a snail. When I started this research and lined up with Bert Sakmann to tackle some of the problems it was of course known from Hodgkin and Huxley that the action potential is a consequence of the permeability changes in nerve membrane but it was not known at this time what the underlying mechanisms are. Now around that time there was another field of research, which was work on artificial membrane. On so-called bimolecular lipid membranes. These were invented by Mueller and Rudin, two American researchers and more or less mimicked the biological membranes. What they found is that these artificial membranes just consisting of lipids were pretty good insulators but when you added tiny amounts of certain substances like the antibiotic gramicidin suddenly you saw, by recording currents across this membrane after applying a voltage, that there was this jumps in current recording. And all the evidence pointed that these gramicidin molecules formed pores in the membranes, so-called ion channels, which would then lead to a conductance. Obviously here these pores would form and decay and the up and down would be the opening and closing of these ion channels. And of course the proposal was that similar ion channels also would be operative in a biological membranes but the proof that was lacking. In fact there were competing hypothesis about carrier mechanism, about phase transitions in the membranes which might produce phenomenal like action potential. But Bert Sakmann and myself set out to prove this channel concept by showing that actually you can record such discontinuous jumps in current on the biological membrane. So just to give you an idea about the order of magnitude the channels of artificial membranes had an amplitude of about one pico-amp. You see here these arrow which should represent currents on a logarithmic scale, I need not go through all of this but just point out that typically the smallest biological currents that could be recorded at the time were on the order of a nano-amp, which is typically the current which is elicited in a muscle when one single quantum of transmitter, one vesicle fuses with the plasma membrane. But the problem was that all these methods for recording currents had an intrinsic noise of about a tenth or two-tenths of an nano-amp and we wanted to record this currents which were hundredfold smaller. So we had to come up with a new idea. The new idea is now known as the so-called Patch Clamp Technique. We used pipettes, measuring pipettes and did not punch through the membrane, as was usual at that time, but tried to place them touchingly on the surface to isolate little patches of membrane and the idea was that if you were lucky there would be a channel we could record the current flowing through this channel through an amplifier. We used a neuromuscular junction and were looking for this ion channels which are activated by the neuromuscular transmitter, which is acetylcholin, and indeed after a little bit of frustration, two or three years, we got recordings of this type. Very clear step-like changes in current elicited by acetylcholin which was added to the recording pipette. A few years later we happened to run across an improvement in the sense that the quality of this recording is very much dependent on the quality of the contact between the recording electrode and the membrane. And we happened to improve this contact about a hundredfold reaching so-called gigaseal conditions which means that the resistance between the inside of the pipette and the outside is in the order of gigaohms and this actually allowed us to record this current in very high precision. Around the same time the molecular biology had advanced, biochemists had been able to isolate membrane proteins, so-called acetylcholine receptors which could be shown to underlie these step like changes in current namely pentameric proteins which had binding sites for acetylcholine. And when this acetylcholine, the neuromuscular transmitter, binds to these molecules they undergo a conformational change, open the pathway through the channel and creating a current. I think that Bert Sakmann in his lecture on Wednesday or Thursday will show a few examples in terms of movies of what you can observe on the oscilloscope when you do this experiment. So let me just point out we were interested in nerve excitability in neuromuscular transmission. We just wanted to show that there are channels and wanted to study these channels. What we did not consider and which came as a surprise to us and actually which made our invention of this technique much more relevant is that channels not only are represented in so-called electrical excitable tissue but fulfil a huge array of different tasks in the whole range of other cells. Basically every cell type we looked at and which we started to look at when we had this method at hand tuned out to contain a specific types of channel which do fulfil all these jobs mainly at the interface between the biochemistry and chemistry and electrical signalling. Particularly in the sensory organs where channels are the real elemental transfusing the end signal from the surround to the central nervous system. What also turned out which we were not aware of is that these channels are prime targets for pharmaca. Just one example here a slide given to me by Harald Reuter an investigation on cardiomyocytes, heart cells, which express a particular type of calcium channel which lets calcium enter the cell to produce the heart contraction. When activated by voltage and under controlled conditions if you have a patch with just a single one of these channels you see upon depolarization this flickering open and close. If you do the same under the influence of a so-called calcium antagonist, a molecule which had been known and had been used as a pharmacon to fight hypertension, these channels are much less willing to open. Their opening is suppressed and therefore there is less calcium influx, less contraction of the heart and particularly also less tone on the blood vessels cells. Okay, another surprise was that defects in ion channels cause a number of hereditary diseases. Some of these are quite common but of course the majority of these diseases which have been characterised in between some fifty to a hundred diseases are quite rare diseases. But still they offer the possibility to learn a lot about human biology. To learn a lot about how very defined changes in a functional molecule in the end turn out to lead to pathological changes. And by now it's clear that about a thousand of the, I think we should say now twenty-thousand genes code for ion channels and transport mechanisms. Okay, so much about ion channels let me guide you one more step on the bottomup approach namely concentrate on synaptic transmission, which is my current field of research. And let me just show once more this summary slide on the basic way how this functions. But let me point out that of course in the end things are much more complex than originally thought. If we look today what biochemists, molecular biologists know about the synapse there about a huge number of molecules engaged in both the pre-synaptic compartment and the post-synaptic compartment. To do this job of synaptic transmission, I mean even though the mechanism is quite complex, neurotransmitter release post-synaptic sensitivity, one could do with just a few of these molecules to make such a complex thing. But in fact we are dealing with about fifteen-hundred interacting proteins here and probably the reason why it is such a complex thing is because of synaptic plasticity. Because of course we not only have to understand how the process takes place but also the nature has to have the means to regulate this synaptic connectivity on all these different time scales in a very complicated way. Okay, so we have been interested in some of the mechanisms of short-term plasticity, the very short forms of changes in synaptic transmission. And our preparation during the last few years has been the so-called Calyx of Held synapse in the auditory pathway second relay station of auditory information. This was already described by Hans Held in 1893, it was re-discovered for electro-physiology by Ian Forsythe who showed that these pre-synaptic terminals are so big that you can use our techniques with the patch pipettes to study in detail the pre-synaptic processes. And Gerard Borst together with Bert Sakmann has shown then that indeed you can do a simultaneously dual whole cell recording which means that you can simultaneously look, record currents and voltage both pre- and post-synaptic, that you can by taking excess with the recording pipette to the inter-cell milieu you can control the ions inside the cell. And you can load the terminals with fluorescent indicators and with so-called caged compounds. This will be one thing which I will come back in a second. So when you record from this synapse you see a large so-called excitatory post-synaptic current, a surge of invert-current as a consequence of the transmitter which has been liberated by the nerve. So this invert-current would use an action potential if it were not voltage clamped, if not our apparatus would hold the potential and the post-synaptic site constant. And the understanding of how this works goes again back to Sir Bernhard Katz who proposed that the release, the signal is proportional to two quantities namely to what's called the number of units available, the number of vesicles ready to be exocytosed when an action potential arrives, times the probability that a given vesicle actually fuses during action potential. Now this was extended by an Australian scientists Vere-Jones in 1966 who said this number of units available is proportional to the number of sites from which such vesicles can be released, times a probability that these sites are occupied. So you have the release to be proportional to the number of sites, times probability these are occupied, times the probability that a vesicle is released. Now, what we can observe on the Calyx of Held are the two most prominent forms of so-called short-term synaptic plasticity namely short-term depression, short-term facilitation. This you can see here when you give a sequence of stimuli at rapid succession you see if you do this here at two millimol calcium which is more or less normal condition that the response depresses, becomes smaller and smaller. Whereas if you do this at a small extracellular calcium concentration the first response is very small, it's different scale here, but subsequent responses are larger. So this is phenomenon of facilitation. Katz's concept gives a simple explanation to this, namely if you take the response proportional to these two kinds of probabilities, of course when you start here the first response releases a large fraction of the units available. So for the second response, the probability of occupancy of a given site is smaller and after a few responses you have more or less exhausted all your store of vesicles and you are left with a very small response. If however, you have a very small first response so the release probability here under these conditions is very small. You hardly change the probability of occupancy during this train and what you then see is that underlying this depression is actually an increase in the release probability for a given vesicle. This is the basis of facilitation. And here in our preparation these two processes superimpose on each other. Other synapses are made up in the sense that you see either only this or that actually also under normal conditions you see the facilitation in cases where release probability of the first pulse is very small. And each type of synapse in different regions of the brain has its own personality in this respect. Now, let me spend two more minutes I hope to just go into another very basic question, which is relevant for synaptic transmission and that is the calcium dependence. Katz already showed that the influx of calcium is the real trigger for the release but up to relatively recently nobody really knew how high this calcium concentration has to rise in order to trigger this event. And how long the duration of such signals and what its properties are. The reason is that of course the concentration of calcium at a given site depends strongly on the neighbourhood relationships. On how far away the calcium source is from the channels which mediate the calcium influx. Since it matters on a length scale of 10µM ~ 100µM culture imaging which is a method available to look at special distribution of calcium signals is not capable of answering this question. So in order to get an idea on how sensitive this release apparatus to calcium is we resorted to so-called caged calcium measurements. We filled the terminal with a compound called calcium compound which is a chelator like EGTA binding calcium but being photo-labile and giving a UV flash we can split this compound. We can liberate calcium and if we make sure that this compound was distributed evenly and also the light is evenly distributed we can elevate calcium uniformly in the cell and having also a calcium indicator dye, a substance which measures calcium concentration, we can then watch these responses to calcium increases, step-like calcium increases to various levels. And we see that if we increase calcium to about 2 micromolar from a pre-calcium level of only one-tenth of the micromolar we see a very, very small response. If we increase calcium to about 6 micromolar we see a response of about the same magnitude as an action potential induced response. So the cause answer is that the release apparatus response to calcium concentrations in the range of 10 micromolar. Of course this is a very course thing and we have to do biophysical modelling in order to get precise answers. But what I showed you establishes that this un-caging allows us to characterise the sensitivity and the speed of response of the release apparatus that from this knowledge we can then by biophysical modelling infer that actually when this calcium increase is only very short-lived we have to have an increase in the region of the vesicles to about 20um. We also can postulate that this increase has to be extremely short-lived because after having characterised the kinetics we know that any longer increase in calcium would lead to much more sluggish and long-lived responses. And further biophysical modelling then shows that actually you need a very tight spatial coordination between the calcium influx and the release sites. So without being able to go into details let me just summarise, we get the speed of the synaptic response by proximity. This provides a need for a very precise ultrastructural organisation of the synapse and this is also one of the reasons why you have these very many specialised molecules in the nerve terminal. So I shouldn't close without mentioning that of course this is the work of the laboratory and the main contributors to what I summarised here are Ralf Schneggenburger a previous post-lab who is now a professor at the EPFL in Lausanne. And Takeshi Sakaba who used to be a post-doc now he has his own group from Japan and from September he will be a professor in Kyoto. Thank you very much.

Erwin Neher on the History of Neurotransmission
(00:00:30 - 00:08:03)


Hormones and Related Messengers
Neurotransmission is a fast process and usually occurs at synapses linking two neurons. If cells require to communicate over longer distance, for example between different parts of the body, the may do so by means of a different group of signalling molecules, the hormones.

In his 2007 Lindau lecture, 1998 Nobel Laureate Ferid Murad familiarized the audience with intercellular, hormonal signalling. He had received the Prize together with Robert Furchgott and Louis Ignarro for the discovery of one of the smallest signalling molecules known: nitric oxide, a molecule consisting of only a nitrogen and an oxygen atom.

Ferid Murad (2007) - Nitric oxide as a messenger molecule and its role in drug development

So after this wonderful start of the meeting, we heard about the Big Bang, and I think the following talk also can be considered that it has been a Big Bang in biomedicine. That was the discovery of nitric oxide. It’s a great pleasure to ask Professor Ferid Murad from Houston, Texas to give his lecture to us here. He won the Nobel Prize in physiology and medicine in 1998. And it was listed for the discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system. Professor Murad. This is my first visit to Lindau. It looks like a very exciting opportunity for the speakers as well as the students and I’m going to enjoy the week, I can assure you. Shortly after the Nobel Prize was announced, my office received numerous phone calls from the local schools in Houston, the high schools, the colleges, asking me to give lectures and meet students and I did that. But the requests really got to be enormous and I couldn’t keep up with it. So I went to the audiovisual television department in the Texas Medical Center and asked if they would help me put a video together. So this morning is going to be movies and films. This department is really an excellent department. They prepare tapes and videos for patients, how do they manage their ileostomy bags, how do they manage their renal shunts for dialysis and so forth. So we got together and put a dialogue together, we exchanged a lot of information back and forth and finally one Saturday morning they showed up at my home and we prepared this video and I think you’ll enjoy it. Now, it was prepared for teenagers but I’ve shown it to 4 year olds, 85 year olds and everybody seems to enjoy it because it’s science in lay language that all of you should understand. So let’s start with the video. Girl 1: Gosh, what’s with all the awards shows? TV: And the winner is... Congratulations. And now I ask you to step forward and receive your Nobel Prize. Girl 1: Nobel Prize? What is that? Girl 2: Beats me. Prof. Dr. Ferid Murad: What? You don’t know what the Nobel Prize is? We have to do something about that. Girl 1: Wow! Girl 2: What’s up with this? Boy: Hey, you’re just coming out of the TV! It’s like aliens. Prof. Dr. Ferid Murad: May I? Girl 1: Yeah sure, why not? So, what’s your story? Prof. Dr. Ferid Murad: Well, my name is Ferid Murad. You can call me Fred. I’m a doctor and researcher and a scientist at the University Of Texas Medical School. And, oh, by the way I got the “Nobel Prize” in medicine in 1998. Girl 2: So, what is that “Nobel Prize” anyway? Prof. Dr. Ferid Murad: Well, the Nobel Prize is one of the greatest awards you can get in the world. Boy: Aha... Prof. Dr. Ferid Murad: It’s recognition from other scientists. Girl 2: So? Prof. Dr. Ferid Murad: Hmm. Well, you get to be on TV all over the world. There’s a big party in Sweden. You even get to meet the King and Queen of Sweden. You get a gold medal. Then of course there is the money. Boy: Money? Girl 1: Party? Girl 2: Royalty? Prof. Dr. Ferid Murad: Yeah. Maybe it would be better if I showed you. May I? Girl 1: Mmm...sure. Prof. Dr. Ferid Murad: Thanks. Scientist on TV: Thanks, Fred. Welcome to my world of science and my laboratory. You know, the Nobel Prize wouldn’t even be around if it wasn’t for...dynamite. Anyway. Alfred Nobel, the Swedish inventor and businessman who invented the Nobel Prizes, was the guy who invented dynamite. Nitro-glycerine is the explosive chemical in dynamite. And even though it was very dangerous, Mr. Nobel figured out a way to contain nitro-glycerine so that it could be put to good use like to build stuff. You could say his discovery rocked the world. But nitro-glycerine has other uses. When Nobel started having heart problems, his doctor actually prescribed nitro-glycerine for his heart. But Nobel said: “No way.” So he blew it. Nobody knew why it worked. But it did. and he shared the 1998 Nobel Prize in Medicine with Dr. Robert Furchgott and Dr. Luis Ignarro for figuring it out. Boy: So you’re the dude that figured out why nitro-glycerine helps peoples’ hearts? Prof. Dr. Ferid Murad: Well, yeah. Girl 2: So? Prof. Dr. Ferid Murad: So what? Girl 2: So why does it work? Prof. Dr. Ferid Murad: We were trying to answer the question to how nitro-glycerine works to help with chest pain. I did experiment, observed the results and collected data. Then I found out if what I thought was right or wrong. Anyway, what I did find was that nitro-glycerine releases nitric oxide and that nitric oxide does a lot of important stuff in the body. Girl 1: So, what is nitric oxide? And what exactly did you figure out that got you this “Nobel Prize”? Prof. Dr. Ferid Murad: Let me show you. Announcer on TV: It protects the heart. It stimulates the brain. It kills bacteria. And it’s a real gas. It’s nitric oxide. No one can say no to NO. Nitric oxide or NO is a simple molecule with two adapts, nitrogen and oxygen. And yes, it’s a colourless, odourless gas. A scientific sensation sweeps the globe. Nitric oxide is everywhere. It’s coming in toxic pollution depletes the ozone layer. It’s even found in car exhaust and cigarette smoke. But from super-menace to superhero. Nitric oxide is also found inside the human body and it helps send very important messages which are not from our sponsors. When blood flows through your blood vessels, the inner lining or endothelium releases nitric oxide. The nitric oxide signals your blood vessel to relax and widen. So what? This in turn lowers blood pressure, the force which the blood exerts on the vessel walls. If your blood vessels make enough nitric oxide, this signals your blood vessel to relax. Then your blood flows on through. No problem. But if blood doesn’t flow through, blood plugs forks. Then... (heart attack). And that’s not all! Scientist on TV: So relaxed blood vessels allow more blood to flow and nitric oxide can have an impact on all different parts of the body. For example, nitric oxide is already saving the lives of babies who are born too early by breathing in very small doses of this gas. It helps their lungs and improves their breathing. And that’s good! In nerve cells nitric oxide can stimulate the brain affecting things like behaviour. Oh behave, baby! As part of the body’s self-defence system nitric oxide defends against tumour cells and bacteria too. It’s amazing stuff. But nitric oxide is no laughing matter and not to be confused with nitrous oxide, better known as laughing gas. Ha-ha... somebody turn of that gas. Boy: So how do you think of all that anyway? Prof. Dr. Ferid Murad: Well, over time I became very interested in how cells talk to each other. But most other scientists didn’t think that was very important. Scientist on TV: Dr. Murad figured out that when cells talk to each other, it’s like one cell sends an e-mail to another cell somewhere in the body. And the e-mail is the gas nitric oxide. The e-mail can break into another cell and take over how the cell works. It may contain a message like instructions for a blood vessel to relax. Or it may contain some other kind of instructions. For example, if the message is being sent to a cancer cell, the nitric oxide may kill the cancer and then self-destruct. Hasta la vista, baby. Nitric oxide in your body affects so many things. It’s like having a worldwide internet system inside your own body. Girl 1: So why did you go into science in the first place? Prof. Dr. Ferid Murad: Well, it’s really a lot of fun to figure out how stuff like this works. And you don’t have to be brilliant to get ahead. You just have to have some goals and be prepared to work very hard. Scientist on TV: As a scientist you get to do something for the first time that nobody else has ever done. And that’s exciting! Sometimes your discovery opens the door to a whole new way of thinking and even more new discoveries. It is really cool! It’s kind of like the “Science Olympics” and the goal is the Nobel Prize. Teams of scientists from around the world compete with each other. It’s fun. Who’s going to finish first? Who’s going to win the prize? Prof. Dr. Ferid Murad: One of you could be a Nobel Prize winner someday. Who knows? Boy: Hey! I have some more questions. Girl 2: Yeah, me too. Girl 1: Well, I guess we have to find out more on our own. Girl 2: Maybe we could check the internet. Boy: Yeah, and look out for science and the Nobel Prize. Prof. Dr. Ferid Murad: They’re gone and they left some popcorn, didn’t they? Yum! I was very fortunate in that I was one of the first MD PhD students in the United States when the program began in Cleveland and I decided that that’s what I wanted to do somehow. I wasn’t sure how I made that decision. I was excited about chemistry and biology. I guess I was confused more than anything else. And said I’m going to try both and see which way to go and I got hooked and I’ve always straddled the fence the rest of my life. My mentors were Earl Sutherland and Ted Rall. They had just discovered cyclic AMP a year before I joined their laboratory. And what an exciting time this was for a young student. My assignment was to figure out how catecholamines, adrenalin regulates cyclic AMP production in tissues and whether it works through the beta adrenergic receptor or the alpha receptor. And that was a pretty straightforward and actually simple assignment. But to see this whole era of cell signalling and messengers evolve was a remarkably exciting time. To see all the hormones that work through these pathways and all the drugs that were evolving that I became hooked on second messenger systems and cell communication. Cell communication is really an old concept, probably first introduced by Pavlov more than 100 years ago. As you recall from your psychology classes Pavlov had a patient with a gunshot wound to his abdomen who developed a gastric fistula to the exterior. And whenever the patient would see or smell food, he would enhance his gastric secretions. Well, Pavlov was clever enough that he developed pouches and fistulas in dogs and showed them food and they too would enhance their gastric secretions. But he decided to condition these dogs by ringing a bell. And ultimately all he had to do was ring a bell and they would enhance their secretion without having to show them food. That told him that the brain was talking to the stomach, cells communicate with each other. Of course we know today that lots of cells and tissues in the body communicate with each other. And this is summarised for you in this cartoon, in this first slide. This represents three different populations of cells that are going to talk to each other. They can be any cells but I’m going to call cell 1 a neuronal cell. It can be a central neuron or a peripheral neuron. Cell 2 I will call an endothelial cell lining a blood vessel and cell 3 a smooth muscle cell in the wall of that blood vessel. Cell 1 wants to talk to cell 2 and also cell 3. And it does it by producing molecules that Earl Sutherland called first messengers. Today we call them hormones, we call them cytokines, we call them growth factors, we call them a variety of things, paracrine substances, autacoids etc. They come in different shapes and sizes and flavours. Some are small amino acids; some are large proteins like the cardiac troponins. The point is they’re released into the interstitial space in the blood stream and they home the body to find its target. It identifies its target cell by the presence in their membrane of a macromolecule that we call a receptor. Sometimes these receptors are inside of the cell, as with steroids and thyroid hormone, but most often they’re transmembrane proteins, integral membrane proteins in the membrane surface. These ligands or first messengers interact with their appropriate cells that only possess those receptors. That’s where the specificity of the reaction occurs. They fit together conformationally, like a key in a lock. And they then only perturb the cells that have the appropriate receptor. The ligand doesn’t necessarily have to enter inside of the cell to cause the cascade of biochemistry to result in some physiologic response. They can just interact with the receptor, it tweaks it and then all of a sudden, voila, there are lots of different intracellular second messengers that accumulate. The first such second messenger was cyclic AMP. What Sutherland and Rall discovered: This is how glucagon and epinephrine regulated glycogen degradation in the liver. We know today that lots of pathways utilise that messenger. We also know that there are other second messengers besides cyclic AMP: cyclic GMP, calcium, diacylglycerol, nitric oxide. And while there are hundreds and hundreds of first messengers, there are a modest number of intracellular second messengers. Perhaps no more than a dozen at present but there will be more, I’m sure, in the future. These second messengers accumulate and carry out the function within the cell that was brought by this first messenger to the cell surface. What is unique about nitric oxide as an intracellular second messenger is that it’s a gas. It’s a free radical with an unshared electron and a very simple molecule. And because it’s uncharged, the physiologic pH, like all the other messengers, it doesn’t go in and out of membranes... it goes in and out of membranes much more readily than the other messengers that often require energy or transporters to do that. So it not only regulates the biochemistry in the cell in which it’s made, nitric oxide, but it can come out and travel a couple of hundred Angstrom, microns, to regulate adjacent cells to produce other second messengers such as cyclic GMP. So it’s a very unique messenger molecule. It’s the only messenger I’m aware of that functions both intracellularly as well as extracellularly. It can be a paracrine substance, a local autacoid. It can also bind with other carriers, glutathione, thiols, albumen, other proteins and be transported a distance to be released again and function therefore as a hormone. No other messengers can do that. Nitric oxide is a very old molecule and I have a theory that it participated also in evolution. It was probably one of the first messenger molecules 3 billion years ago, as cells began to communicate with each other. But I won’t get into that story because time doesn’t permit. But today we know that it’s very important as a pollutant. And this is when it became popular about 50 or 60 years ago when it was apparent that all fossil fuels when combusted with oxygen produced a family of nitrogen oxides, NO, NO2, N2O3 etc. All of these nitrogen oxides will interact with ozone and deplete the ozone layer and are responsible for global warming along with the greenhouse gases. But I’m going to tell you that not only is the nitric oxide a pollutant but it’s the mechanism of action of some very important cardiovascular drugs and it’s a very important messenger molecule in the body. And this is just a partial list of the biology that it regulates. And the list is much, much longer than this. It includes muscle relaxation, platelet aggregation, penile erection, killing of parasites and microorganisms and the list goes on and on. We’ll come back to some of that shortly. In 1963, 7 years after the discovery or 6 years after the discovery of cyclic AMP, a couple of chemists discovered for the first time cyclic GMP, another cyclic nucleotide, a cousin of cyclic AMP. All you do is shift the amino group on the purine ring and you have a different structure. They administered inorganic P32 phosphate to rats, harvested the urine and found two major organic phosphates in urine, cyclic AMP, the other being cyclic GMP. That was the first demonstration of cyclic GMP as a natural product. That stimulated a few laboratories to become interested in cyclic GMP, to look for enzymes that made it, enzymes that hydrolysed it etc. And the story began to evolve in the late 1960s, early 1970s just as I was finishing my training and getting ready to join the faculty at the University of Virginia. We know today that there is a family of enzymes called guanylyl cyclases and there are at least seven or eight members of this family and we think there will be even more with splice variants that we’re now isolating. All of these enzymes convert GTP to cyclic GMP and pyrophosphate. The reaction is very analogous to add an OH cyclase that converts ATP to cyclic AMP and pyrophosphate. In fact the catalytic domains of all of the cyclases are homologous with the change of one or two amino acids, one enzyme can make the other nucleotide. The cyclic GMP can be inactivated by a family of phosphodiesterases; there are at least 10 or 11 members in this gene family. And they hydrolyse the phosphodiester bond to convert the cyclic nucleotide to the corresponding monophosphate, either 5’ GMP or 5’ AMP and it becomes inactive. As you will hear later in the week from Doctor Fischer, many of the intracellular second messengers, cyclic AMP, cyclic GMP, diacylglycerol, calcium etc., often activate a protein kinase that then phosphorylates a variety of protein substrates by transferring the gamma phosphate from ATP to a serine-threonine residue or perhaps a tyrosine residue. There are I’m told now as many as 500 or 600 protein kinases. So while the concept of cell communication is pretty simple, a ligand or hormone regulates the second messenger production which then regulates the protein kinase which phosphorylates a protein, the problem is the matrix gets pretty hairy when you consider all the cyclases, the diastasis, the protein kinases and the numerous proteins that can be phosphorylated. When these proteins are phosphorylated, as you’ll hear from Doctor Fischer, their conformation changes. If they’re structural proteins they can influence motility and other processes, contractility. If they’re enzymes they can be activated or inhibited. So as this story was beginning to unfold with cyclic GMP, I decided to desert cyclic AMP and switch my interest to cyclic GMP. As a new young faculty member in the early 1970’s the cyclic AMP field in my opinion was becoming rather crowded, large groups of people around the world. And I didn’t think that I could compete with all these huge laboratories and cyclic GMP was evolving and I said I’m going to go in this new direction. And I wanted to address two questions. We knew that a couple of hormones could increase cyclic GMP accumulation in a couple of tissues, acetylcholine in heart preparations, prostaglandins in vascular preparations. But we didn’t know the molecular coupling mechanism. If we understood ligand binding to the receptor and the coupling to the guanylyl cyclase to make cyclic GMP, we could presumably influence that coupling process with various chemicals and drugs to potentiate a hormone response or block it and maybe come up with some novel therapies for various endocrine diseases. That was quite rational. The second is, although cyclic GMP was a natural product, we had no idea what it did or what its function was. So we began our studies by looking at the enzyme in some detail that made cyclic GMP and the first big surprise was that there wasn’t just a single enzyme but a couple of enzymes. The enzyme activity was in soluble fractions as well as particulate fractions. The kinetic properties of this isoforms was different. I suspected there would be isoforms. I had thought to myself: If there are different compartments and isoforms of guanylyl cyclase, perhaps they’re regulated by different groups of hormones, wouldn’t that be a fun project to sort out. And a lot of that all turned out to be true intuition at the time. Well, to prove that we were dealing with isoforms was going to take a lot of work. We had to purify these soluble particulate activities, clone them, express them, restudy them. We did that, it took us 12 or 15 years of work to do all that. But initially I took a shortcut. I said the cooperativity of the particulate isoform, whereas the typical Michaelis-Menten kinetics of the soluble could be artifactual because these were crude preparations that possessed nucleotidases, phosphatases, phosphodiesterases. So we created a cocktail and added it to our incubations to inhibit all these competing pathways. We made a cocktail pyrophosphate, fluoride, azide, hydroxylamine, sodium nitrite, methylxanthines etc. And quite accidentally, as science often is the case, we found compounds that activated the enzyme. While our goal was to figure out hormonal regulation, we couldn’t do that because once we disrupted tissues, the hormone coupling was lost. Hormones no longer activated extracts of the enzyme. But the small molecules now would activate and maybe they could be our surrogates for understanding hormonal regulation. Much as fluoride was a valuable resource for understanding hormonal regulation of adenylate cyclase. The activators were azide, hydroxylamine and sodium nitrite. And to make a long story short, the effects of the azide were oxygen dependent, enhanced with thiols, had a time lag before the rate of the reaction became maximal. They were tissue specific because tissues possessed inhibitors and activators of the pathway. And we were convinced that these activators were precursors or prodrugs being converted to something else in our incubation. And it became quite a mystery story for several years to figure out what that activator was going to be. We put azide and hydroxylamine and nitrite on cell cultures in tissues. And indeed they would elevate cyclic GMP levels and would also activate the enzyme and extracts. So they would work in both systems. One of the tissues, again fortunately, that we were working with was tracheal smooth muscle. As a student I knew that cyclic AMP relaxed smooth muscle, vascular and airway smooth muscle. And I thought cyclic GMP might antagonise cyclic AMP. So we prepared a smooth muscle preparation that was relatively homogenous in order to do some biochemistry and compare it with a physiologer. We put azide on those smooth muscle preparations, the cyclic GMP levels went up, but the muscle didn’t contract, it relaxed. The opposite of what we expected. The dose response curves, the time courses, everything told us that azide was a smooth muscle relaxant, mediating its effects through cyclic GMP and that’s how it worked. And that was the first physiologic function of cyclic GMP. We said: If these drugs cause relaxation and elevate G, what do the other smooth muscle relaxants do? Nitro-glycerine, which had been around for 100 years, nitroprusside? A lot of popular drugs in the cardiovascular intensive care units. They decreased after load, lowered blood pressure in patients with infarction and so forth. We put them on our preparations, they relaxed as expected. The surprise was that they elevated cyclic GMP levels. So now we had a family of compounds that we called nitrovasodilators that were capable of activating guanine cyclase in cell free preparations and elevating cyclic GMP levels in various tissues. These compounds included hydroxylamine, azide, sodium nitrite, some hydrazines, nitro-glycerine, nitroprusside, nitrosoureas, nitrosamines and a long list of other compounds, all NO donors. They all have nitrogen. Some are converted spontaneously based on redox and pH incubations. Some are converted insomatically, azide requires catalase conversion. And we reasoned that the intermediate for all of these prodrugs or precursors had to be nitric oxide. And the reason we thought it was nitric oxide is because azide’s tissue specificity was in part influenced by the presence of haemoglobin and myoglobin in our tissue extracts. These were inhibitors. We knew from the literature that NO had a very high affinity for the heme prosthetic group of haemoglobin, myoglobin. So we reasoned that all of these prodrugs or precursors were converted to nitric oxide. When we generated nitric oxide in the fume hood chemically, sodium nitrite, ferrous sulphate, sulphuric acid, ventilated the gas into our incubations, every preparation was activated. What an exciting period that was. The first demonstration that a free radical could activate an enzyme. And I thought that would be interesting chemistry. Lots of folks were sceptical. They all thought of free radicals and the nitric oxides as pollutants and toxic materials and sure enough it activated. Some people argued: Murad, you’re inhibiting an inhibitor, you’re disinhibiting in your crude preparations. So we were obliged to purify the enzyme to homogeneity and when we did the concentration the nitric oxide required to activate kept becoming lower, lower, lower as we moved scavengers and sinks from our incubation. Transition metals, thiols, proteins and sucked it up, you know. Nanomolar concentrations of NO would activate the enzyme and the Vmax would increase 200- to 400-fold, as it would lower the Km for GTP. So we realised that we had figured out the mechanism of action of the nitrovasodilators, such as nitro-glycerine that had been used clinically for 100 years. As a pharmacologist, when you find an exogenous material that does something in a biological system, you should ask yourself: Is it mimicking a natural pathway that’s already there? And is it working through a similar mechanism? So I proposed in some review articles in the late ‘70s that perhaps the effects of various hormones to increase cyclic GMP levels were because they were increasing the production of nitric oxide from some endogenous precursor, perhaps by altering some redox pathway. We couldn’t prove that hypothesis because the technology was not there to measure nitric oxide in its oxidation products at animals or concentrations back in the 70’s. There were crude colorimetric assays. So to prove this took another seven or eight years of technology development before we and others were able to prove that it turned out to be the case. But we expected that nitric oxide was going to be an intracellular second messenger and that was heresy. And it was enough to show that it activates... a free radical activates an enzyme, now you're saying a free radical is a second messenger and it turned out to be true, we turned out to be right. And basically that’s the reason we went to Stockholm. Shortly after this Robert Furchgott, a vascular pharmacologist in New York, showed for the first time that a group of agents would relax vascular segments in the organ bath in the laboratory. Agents such as acetylcholine, histamine, bradykinin that were known to be hypotensive in man or animals had always failed to cause relaxation in the laboratory. He found, if he preserved the integrity of the endothelium in his blood vessel preparations, they would cause relaxation. And that was an exciting turn of events in 1980. I heard him present this work, he said that these substances caused the release of a factor from the endothelium which he called endothelium-derived relaxant factor, or EDRF, and had a half-life of only several seconds. I said: Bob, this is a reactive species, maybe a free radical, maybe it works in cyclic G, maybe it’s going to be related somehow to NO. Let’s figure this out. Well, we moved to Stanford, Bob went to New York, his wife developed breast cancer and that collaboration never took place. But a couple of years later we became impatient and moved on ourselves and showed that that hypothesis was in fact the case. This is a blood vessel precontracted with norepinephrine. And then after five or seven minutes we introduced an endothelial dependent vasodilator, acetylcholine, bradykinin, ATP ionophore, histamine etc. And if the endothelium is present, cyclic GMP levels increased within seconds, returned to basals and this is followed by relaxation in the muscle. But that only occurs if the endothelium is intact, if there’s no endothelium there’s no increase in cyclic G, no relaxation. So we knew by the early 1980s there were now two pathways converging on guanylyl cyclase to regulate cyclic GMP synthesis. The NO donors in the endothelial dependent vasodilator pathway. We spent a couple of years showing that this resulted in the activation of protein kinase G in vascular smooth muscle preparations. We then chased the phosphorylation of a variety of proteins with P32 incorporation. And to put all of the story together for you, it’s summarised on this slide. This cartoon represents a blood vessel with the endothelial lining on the left and the smooth muscle compartment on the right and in red are three categories of vasodilators that work by enhancing cyclic GMP production. The nitrovasodilators that are converted spontaneously based on pH oxygen tension etc. or the enzymatically converted, such as nitro-glycerine, to nitric oxide which activates the soluble guanylyl cyclase. Soluble guanylyl cyclase is a heterodimer with an alpha-beta-subunit and the beta-subunit has a heme prosthetic group at the histamine 105 position. The iron in that heme has to be ferrous, NO binds to the ferrous, changes the liganding of the heme to the beta-subunit which then talks to the catalytic domain down at the C-terminal, resulting in activation of the enzyme. The cyclic GMP activates a cyclic G dependent protein kinase. There are a couple of these, soluble and particulate that phosphorylate numerous proteins. These proteins regulate the concentration of cytosol calcium by altering distribution within the cell from stores or through channels in the membrane. And we know that the myosin light-chain kinase is a calcium calmodulin dependent enzyme. So when you decrease calcium, you decrease the kinase, myosin light-chain accumulates in the dephosphorylated state. If you take actin and myosin filaments, when you phosphorylate the myosin light-chain, they slide together for contraction. You dephosphorylate they slide apart and you get relaxation. Since this work it’s been shown that cyclic GMP also regulates the phosphatase activity perhaps. The endothelial dependent vasodilators do exactly the same thing; however, the receptors for these endothelial dependent vasodilators are only on the endothelial cells, not the smooth muscle cells. That’s why patients with arthrosclerosis and cardiovascular disease get direct acting NO donors, nitro-glycerine, nitroprusside etc. They don’t get endothelial dependent vasodilators because their endothelium is abnormal. And I’ll tell you more about that in a moment. But they produce EDRF, which we know is nitric oxide, to work through the same pathway. A third category are the atriopeptins. It was found some years ago by deBold that the granules and atria possess a peptide called atrial natriuretic factor and we found that that factor, atriopeptins and the family of these, activate the particulate isoforms of guanylyl cyclase and there are several of these. It turns out the particulate cyclases are receptor cyclases. For atriopeptins, guanylyl, e-coli enterotoxin etc. And by elevating cyclic G, they too will cause relaxation. Now, you can relax smooth muscle in other ways with agents that alter calcium, agents that increase cyclic AMP, phosphodiesterase inhibitors etc. These are the mechanisms in which you cause relaxation by enhancing cyclic GMP production. But you can start combining this information now to create novel combination therapies for various cardiovascular diseases and I’ll show you how to do that in a moment. There was some other very important observations along the way and I’m going to have to skip them. But it became apparent by the mid and late 1980s that there had to be a pathway that was making nitric oxide. People were finding nitride and nitrate in their condition media, people were finding arginine activation of the pathway. And it turns out indeed there’s a family of enzymes called nitric oxide synthases, very ubiquitous, found in most tissues. And there are 3 isoforms, initially called neuronal NOS, inducible NOS, endothelial NOS, the tissues which they were first purified and characterised. Because they’re so ubiquitous, I’ve suggested we call them NOS-1, 2 and 3 in the chronological order in which they were first characterised, purified, cloned. Very homologous, about 50% to 60% homology with each other. Some are soluble, some are particulates, lots of opportunity for post-translational modifications. Some are acylated with polmitate or myristate; many of them are phosphorylated by various kinases. A very complicated family. Normally this NOS-2 or inducible NOS is not found in tissues. You don’t see transcript or protein unless there’s been an inflammatory insult. So it’s become a biomarker of inflammation. You can enhance its production by anything that turns on the NF-kappaB pathway, endotoxin, pro-inflammatory cytokines, IO1, interfering gamma etc. And you make a lot of enzyme which is a high output production stage for nitric oxide that will participate in a lot of cytoxic and inflammatory processes. All of these enzymes catalyse this reaction. They oxidise the guanidino nitrogen of arginine. They oxidise the turtle guanidino nitrogen to an intermediate hydroxy arginine, further oxidised NO and citrulline. The enzymes are only active as homodimers. They have a heme prosthetic group. They require NADPH oxygen flavines, heme as a prosthetic group. Very complicated. If the bioptron is oxidised to the dihydro, it no longer makes NO but the enzyme now makes super-oxide, one of the worse things you can do in nitric oxide biology. So let me put this pathway together for you. We know now that various hormones and ligands will interact with their appropriate receptors to regulate various co-factors for the nitric oxide synthase pathways to convert arginine to nitric oxide and cytroline. This then activates soluble guanylyl cyclase to make cyclic GMP which activates a kinase and the cascade goes on to cause some processor event. We can influence this pathway in a variety of ways now with pharmacological tools. We have compounds that are receptor agonist and antagonist. We can modify the availability of the cofactors. We have compounds that are arginine analogues or guanidino analogues to block and inhibit the nitric oxide synthases. Some of these are in clinical trials for septic shock and inflammatory diseases. We can scavenge the NO not only with haemoglobin and monoglobin but also with various thiols that are also in clinical trials. There are compounds available that activate the cyclase without NO. We have a mutant enzyme that doesn’t require NO for gene therapy some day we think. You can also enhance the activation by NO or another gas, carbon monoxide, which is a weak activator in the presence of compounds, and there are inhibitors available as well. So we have tools now to begin to approach this system. But the system is a little bit more complicated than that, it always is. When you make nitric oxide, what it wants to do is activate guanylyl cyclase to make cyclic G, to inhibit platelet aggregation, to act as a neurotransmitter, to influence smooth muscle relaxation, all the beneficial sorts of things. However it can get oxidised to nitrite nitrate, it can form complexes with other transition metals. It will form complexes with various thiol groups and proteins. More than 100 proteins get nitrosated on the cysteine residues and one of the important pathways is the interaction of nitric oxide with superoxide anion. This reaction is almost diffusion limited. So whenever you’ve got either in contact with one another, that’s the reaction that will take place and it acts as a steal or a sink to pull it away from the cyclic G pathway. And you make peroxynitrite, which is even more toxic than nitric oxide or superoxide. Peroxynitrite will nitrate protein residues adjacent to the hydroxyl and tyrosines, perhaps sterically inhibiting or interfering with tyrosine kinase phosphorylation in some cases. So maybe the two systems are talking to each other. The tyrosine kinase pathway, the NO pathway, perhaps. There’s been very little evidence in vivo showing that, most of the evidence so far as been in vitro. There is an important disease called endothelial dysfunction. Many of us, certainly the older folks in this audience, have endothelial dysfunction, maybe not so many of the younger people yet. It occurs with hypertension, atherosclerosis, diabetes, tobacco use and perhaps obesity. In all of these disorders there’s oxidated stress, the formation of reactive oxygen species, hydrogen peroxide, superoxide anion, hydroxyl radical and the tissue of blood vessels don’t make sufficient quantities of nitric oxide, for a variety of reasons and I’m not going to go through all the reasons. But the net outcome is your blood vessels don’t make enough NO. They tend to be vasoconstricted, you accelerate the atherosclerotic process. You diminish perfusion in diabetic ulcers and lesions. So it’s a vicious cycle, as you get more constricted more reactive oxygen species, less NO. It gets worse and worse. So you’ve got to interrupt it, you’ve got to treat the underlying disease and perhaps come along with some new approaches, perhaps arginine supplementation to make more NO, antioxidants to get rid of the reactive oxygen species. And there are a lot of clinical, a lot of annual studies to support that. There are some clinical studies. The clinical studies have been very controversial. Almost through here, another slide. The nitric oxide field is gone absolutely bananas. Our first paper on the biological effects was in 1977. Almost as many as cyclic AMP, more than G proteins and a lot of other things. It’s incredible. That is about 6,000 to 8,000 papers per year, in the last 5 to 10 year period. That’s 20 to 30 papers a day. There’s no one in the world to keep up with it. It’s very frustrating for all of us in the field. But it’s also very exciting to see this interest and enthusiasm. I’ve created a partial list for you, this slide and the next, to show you where this field is going to go I think in the next decade with regard to a novel drug development and how you can take these biochemical molecular targets that we and others have described and turn them around now in high throughput screens and other assays to find chemical leads to make new therapeutics. We know, as I said, that NO is participating as a neurotransmitter in the brain, in some regions of the brain, both centrally and peripherally. If you make a NOS-1 knockout mouse and induce a cerebral infarct, the size of the infarct is smaller. If you make a NOS-3 knockout mouse, induce an infarct, the infarct is bigger. If you do a double knockout, NOS-1 and NOS-3, the mouse is not very smart anymore, it can’t get through the water maze just to find the flow. So the participations of nitric oxide is transmitters in various regions, also with memory and we certainly haven’t figured all that out. We know it plays a role in fluid production and re-absorption, in the eye for glaucoma, retinal neuronal degeneration. We talked a lot about vasodilation; pulmonary hypertension was mentioned in the movie. It’s been exciting to see that develop. Premature babies maintained in in vivo circulation, they don’t need to profuse their lungs because they get their oxygen from the placenta from their mother. If they’re born prematurely, their blood vessels are constricted, their lungs are not developed, they need surfactant, you need to dilate those blood vessels and they shunt right to left, so they’re hypoxic babies. If you give them a little nitric oxide in the nasal catheter, they dilate the vessels, they don’t shunt quite as much, they become oxygenated. There’s been a marvellous improvement to eliminate the need for ECMO therapy, which is rather nasty. You all know about Viagra. It came out of this research, not us but we developed the concepts. We used to joke in the lab, we’ll fill condoms with vasodilators and NO donors and PD inhibitors, they work. Viagra is an inhibitor of the type 5 phosphodiesterase which is found predominantly in blood vessels, both in the corpus cavernosum as well as the systemic vessels. This is why patients who take Viagra and now take NO donors, nitro-glycerine, can get severe hypertension, arrhythmia, strokes etc. So one has to be careful, it’s not a street drug, it can get you in trouble. It participates in wound healing and angiogenesis, atherosclerosis. If you look at exhalation of NO in your breath, it’s a wonderful marker of the severity of asthma. The more inflammation in your lungs, the more induction of NOS-2, the more NO you make, the more NO in your breath, so the clinician can have you breathe into a bag or tube and if the NO is elevated, he knows that your asthma is out of control and he’ll readjust therapy. It’s been very useful. There are clinical studies for septic shock, platelet aggregation. We know that NO regulates genes, both up and down from micro array studies on cells. We’ve become very interested in stem cell biology, the last several years we know that nitric oxide and cyclic G influence embryonic human and mouse stem cell proliferation and differentiation. We think we can force cells into different lineages by playing games with NO and cyclic G. And there are other applications, this is only a partial list, we can’t do everything. Thank you very much.

Ferid Murad on the History of Intercellular Signalling
(00:13:34 - 00:17:23)

As for the vitamins, the discovery of hormones began not earlier than 1902, making hormone research “highly susceptible” to Nobel Prizes. However, while in that year, the Brits Wiliam Bayliss and Ernest Starling did discover secretin, the first hormone ever identified, and while the two scientists even coined the term “hormone” in 1905, they never received the Nobel Prize.

Secretin is not a small, but a “large” protein molecule. It is released from the small intestine upon the arrival of food from the stomach. The hormone then triggers the release of digestive juices by the pancreas, a classical example of between-organ signalling. And, importantly, a process not involving the brain, as previously assumed. Curiously, Ivan Pavlov, introduced as the father of chemical signalling by Ferid Murad above, received the unshared 1904 Nobel Prize in Physiology or Medicine for a theory of digestion, which still put the brain at the centre of the physiology of gastric secretion. The award was given, although Pavlov’s theory had been proven incorrect by Bayliss and Starling two years before. A controversial decision.

Another “large” protein hormone tied to a much discussed Nobel Prize is insulin. The story of its discovery, and a description of the controversy around the associated 1923 Nobel Prize in Physiology or Medicine, may be found on the website of the Nobel Foundation [10].

In 1939, the first Nobel Prize involving small molecule hormones was given to the German Adolf Butenandt. The chemist received half of the award for his work on sex hormones. And it was hard work indeed. Butenandt and his team had processed thousands of litres of urine, collected from pregnant women, in order to isolate a couple of milligrams of the sex hormone oestrone. Butenandt also isolated androsterone from men’s urine and progesterone from pig ovaries, determining the structure of the compounds. Jointly with his co-recipient, the Swiss-Croatian Leopold Ruzicka, he eventually established that several sex hormones may be synthetically derived from a common precursor, cholesterol, the first known steroid.

In 1950, three scientists received the Nobel Prize for Physiology or Medicine for a similar feat. Edward Kendall and Philip Hench, both from the USA, and the Swiss-Polish Tadeus Reichstein were rewarded for their work on the hormones of the adrenal cortex. It included the isolation and structural characterization of about 29 small molecules found in the adrenal cortex and some early studies on their biological effects. The best known of these compounds is cortisone, which was later shown by Hench to be an effective anti – rheumatic agent. Like the sex hormones, the hormones of the adrenal cortex belong to the chemical group of steroids. At the 1954 Lindau Nobel Laureate Meeting, Reichstein told the story of their discovery:

Tadeus Reichstein on the Hormones of the Adrenal Cortex (in German)
(00:00:15 - 00:07:29)

It may be added that, in 1934, Reichstein also developed a technical procedure for the large-scale production of vitamin C. The so-called Reichstein process relies on the use of the synthetic power of bacteria and has dominated industrial vitamin C production for decades. It is still used today in modified form [9].

Back in the world of hormones, the next Nobel Prize was awarded in 1971 to Earl Sutherland "for his discoveries concerning the mechanisms of the action of hormones". Sutherland had elucidated the concept of first and second messengers (introduced by Ferid Murad above) and discovered the important (small) second messenger molecule cyclic AMP. Unfortunately, Sutherland never came to Lindau. Six years after his distinction, Roger Guillemin and Andrew V. Schally shared one of the 1977 Nobel Prizes in Physiology of Medicine "for their discoveries concerning the peptide hormone production of the brain". They had investigated how the brain controls certain hormone producing glands, like the thyroid, and found that it uses yet another set of “large” protein hormones to do so.

The next Nobel Prize for small molecule signalling came in 1982 and was awarded jointly to Sune Bergström, Bengt Samuelsson and John Vane "for their discoveries concerning prostaglandins and related biologically active substances". Prostaglandins are locally acting messenger molecules affecting only those cells in the vicinity of the signalling cell (hence, they formally do not belong to the class of hormones). They exert an extremely wide variety of strong physiological effects and act in practically every organ, tissue, and cell of our bodies [11]. Prostaglandins trigger the constriction and relaxation of muscle tissue, induce labour and play important roles in pain, fever and inflammation, to name only a few examples. Drugs suppressing prostaglandin biosynthesis, such as aspirin or ibuprofen, are commonly used as anti-inflammatory agents, while the prostaglandins themselves have found several clinical applications including the induction of childbirth or early abortions [11].

This brings us to the last Nobel Prize given for the discovery of a small signalling molecule so far. It was given to Robert Furchgott, Louis Ignarro and Murad, who had elucidated nitric oxide signalling. Murad, who introduced this subsection, told the story of nitric oxide later in his talk:

Ferid Murad (2007) - Nitric oxide as a messenger molecule and its role in drug development

So after this wonderful start of the meeting, we heard about the Big Bang, and I think the following talk also can be considered that it has been a Big Bang in biomedicine. That was the discovery of nitric oxide. It’s a great pleasure to ask Professor Ferid Murad from Houston, Texas to give his lecture to us here. He won the Nobel Prize in physiology and medicine in 1998. And it was listed for the discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system. Professor Murad. This is my first visit to Lindau. It looks like a very exciting opportunity for the speakers as well as the students and I’m going to enjoy the week, I can assure you. Shortly after the Nobel Prize was announced, my office received numerous phone calls from the local schools in Houston, the high schools, the colleges, asking me to give lectures and meet students and I did that. But the requests really got to be enormous and I couldn’t keep up with it. So I went to the audiovisual television department in the Texas Medical Center and asked if they would help me put a video together. So this morning is going to be movies and films. This department is really an excellent department. They prepare tapes and videos for patients, how do they manage their ileostomy bags, how do they manage their renal shunts for dialysis and so forth. So we got together and put a dialogue together, we exchanged a lot of information back and forth and finally one Saturday morning they showed up at my home and we prepared this video and I think you’ll enjoy it. Now, it was prepared for teenagers but I’ve shown it to 4 year olds, 85 year olds and everybody seems to enjoy it because it’s science in lay language that all of you should understand. So let’s start with the video. Girl 1: Gosh, what’s with all the awards shows? TV: And the winner is... Congratulations. And now I ask you to step forward and receive your Nobel Prize. Girl 1: Nobel Prize? What is that? Girl 2: Beats me. Prof. Dr. Ferid Murad: What? You don’t know what the Nobel Prize is? We have to do something about that. Girl 1: Wow! Girl 2: What’s up with this? Boy: Hey, you’re just coming out of the TV! It’s like aliens. Prof. Dr. Ferid Murad: May I? Girl 1: Yeah sure, why not? So, what’s your story? Prof. Dr. Ferid Murad: Well, my name is Ferid Murad. You can call me Fred. I’m a doctor and researcher and a scientist at the University Of Texas Medical School. And, oh, by the way I got the “Nobel Prize” in medicine in 1998. Girl 2: So, what is that “Nobel Prize” anyway? Prof. Dr. Ferid Murad: Well, the Nobel Prize is one of the greatest awards you can get in the world. Boy: Aha... Prof. Dr. Ferid Murad: It’s recognition from other scientists. Girl 2: So? Prof. Dr. Ferid Murad: Hmm. Well, you get to be on TV all over the world. There’s a big party in Sweden. You even get to meet the King and Queen of Sweden. You get a gold medal. Then of course there is the money. Boy: Money? Girl 1: Party? Girl 2: Royalty? Prof. Dr. Ferid Murad: Yeah. Maybe it would be better if I showed you. May I? Girl 1: Mmm...sure. Prof. Dr. Ferid Murad: Thanks. Scientist on TV: Thanks, Fred. Welcome to my world of science and my laboratory. You know, the Nobel Prize wouldn’t even be around if it wasn’t for...dynamite. Anyway. Alfred Nobel, the Swedish inventor and businessman who invented the Nobel Prizes, was the guy who invented dynamite. Nitro-glycerine is the explosive chemical in dynamite. And even though it was very dangerous, Mr. Nobel figured out a way to contain nitro-glycerine so that it could be put to good use like to build stuff. You could say his discovery rocked the world. But nitro-glycerine has other uses. When Nobel started having heart problems, his doctor actually prescribed nitro-glycerine for his heart. But Nobel said: “No way.” So he blew it. Nobody knew why it worked. But it did. and he shared the 1998 Nobel Prize in Medicine with Dr. Robert Furchgott and Dr. Luis Ignarro for figuring it out. Boy: So you’re the dude that figured out why nitro-glycerine helps peoples’ hearts? Prof. Dr. Ferid Murad: Well, yeah. Girl 2: So? Prof. Dr. Ferid Murad: So what? Girl 2: So why does it work? Prof. Dr. Ferid Murad: We were trying to answer the question to how nitro-glycerine works to help with chest pain. I did experiment, observed the results and collected data. Then I found out if what I thought was right or wrong. Anyway, what I did find was that nitro-glycerine releases nitric oxide and that nitric oxide does a lot of important stuff in the body. Girl 1: So, what is nitric oxide? And what exactly did you figure out that got you this “Nobel Prize”? Prof. Dr. Ferid Murad: Let me show you. Announcer on TV: It protects the heart. It stimulates the brain. It kills bacteria. And it’s a real gas. It’s nitric oxide. No one can say no to NO. Nitric oxide or NO is a simple molecule with two adapts, nitrogen and oxygen. And yes, it’s a colourless, odourless gas. A scientific sensation sweeps the globe. Nitric oxide is everywhere. It’s coming in toxic pollution depletes the ozone layer. It’s even found in car exhaust and cigarette smoke. But from super-menace to superhero. Nitric oxide is also found inside the human body and it helps send very important messages which are not from our sponsors. When blood flows through your blood vessels, the inner lining or endothelium releases nitric oxide. The nitric oxide signals your blood vessel to relax and widen. So what? This in turn lowers blood pressure, the force which the blood exerts on the vessel walls. If your blood vessels make enough nitric oxide, this signals your blood vessel to relax. Then your blood flows on through. No problem. But if blood doesn’t flow through, blood plugs forks. Then... (heart attack). And that’s not all! Scientist on TV: So relaxed blood vessels allow more blood to flow and nitric oxide can have an impact on all different parts of the body. For example, nitric oxide is already saving the lives of babies who are born too early by breathing in very small doses of this gas. It helps their lungs and improves their breathing. And that’s good! In nerve cells nitric oxide can stimulate the brain affecting things like behaviour. Oh behave, baby! As part of the body’s self-defence system nitric oxide defends against tumour cells and bacteria too. It’s amazing stuff. But nitric oxide is no laughing matter and not to be confused with nitrous oxide, better known as laughing gas. Ha-ha... somebody turn of that gas. Boy: So how do you think of all that anyway? Prof. Dr. Ferid Murad: Well, over time I became very interested in how cells talk to each other. But most other scientists didn’t think that was very important. Scientist on TV: Dr. Murad figured out that when cells talk to each other, it’s like one cell sends an e-mail to another cell somewhere in the body. And the e-mail is the gas nitric oxide. The e-mail can break into another cell and take over how the cell works. It may contain a message like instructions for a blood vessel to relax. Or it may contain some other kind of instructions. For example, if the message is being sent to a cancer cell, the nitric oxide may kill the cancer and then self-destruct. Hasta la vista, baby. Nitric oxide in your body affects so many things. It’s like having a worldwide internet system inside your own body. Girl 1: So why did you go into science in the first place? Prof. Dr. Ferid Murad: Well, it’s really a lot of fun to figure out how stuff like this works. And you don’t have to be brilliant to get ahead. You just have to have some goals and be prepared to work very hard. Scientist on TV: As a scientist you get to do something for the first time that nobody else has ever done. And that’s exciting! Sometimes your discovery opens the door to a whole new way of thinking and even more new discoveries. It is really cool! It’s kind of like the “Science Olympics” and the goal is the Nobel Prize. Teams of scientists from around the world compete with each other. It’s fun. Who’s going to finish first? Who’s going to win the prize? Prof. Dr. Ferid Murad: One of you could be a Nobel Prize winner someday. Who knows? Boy: Hey! I have some more questions. Girl 2: Yeah, me too. Girl 1: Well, I guess we have to find out more on our own. Girl 2: Maybe we could check the internet. Boy: Yeah, and look out for science and the Nobel Prize. Prof. Dr. Ferid Murad: They’re gone and they left some popcorn, didn’t they? Yum! I was very fortunate in that I was one of the first MD PhD students in the United States when the program began in Cleveland and I decided that that’s what I wanted to do somehow. I wasn’t sure how I made that decision. I was excited about chemistry and biology. I guess I was confused more than anything else. And said I’m going to try both and see which way to go and I got hooked and I’ve always straddled the fence the rest of my life. My mentors were Earl Sutherland and Ted Rall. They had just discovered cyclic AMP a year before I joined their laboratory. And what an exciting time this was for a young student. My assignment was to figure out how catecholamines, adrenalin regulates cyclic AMP production in tissues and whether it works through the beta adrenergic receptor or the alpha receptor. And that was a pretty straightforward and actually simple assignment. But to see this whole era of cell signalling and messengers evolve was a remarkably exciting time. To see all the hormones that work through these pathways and all the drugs that were evolving that I became hooked on second messenger systems and cell communication. Cell communication is really an old concept, probably first introduced by Pavlov more than 100 years ago. As you recall from your psychology classes Pavlov had a patient with a gunshot wound to his abdomen who developed a gastric fistula to the exterior. And whenever the patient would see or smell food, he would enhance his gastric secretions. Well, Pavlov was clever enough that he developed pouches and fistulas in dogs and showed them food and they too would enhance their gastric secretions. But he decided to condition these dogs by ringing a bell. And ultimately all he had to do was ring a bell and they would enhance their secretion without having to show them food. That told him that the brain was talking to the stomach, cells communicate with each other. Of course we know today that lots of cells and tissues in the body communicate with each other. And this is summarised for you in this cartoon, in this first slide. This represents three different populations of cells that are going to talk to each other. They can be any cells but I’m going to call cell 1 a neuronal cell. It can be a central neuron or a peripheral neuron. Cell 2 I will call an endothelial cell lining a blood vessel and cell 3 a smooth muscle cell in the wall of that blood vessel. Cell 1 wants to talk to cell 2 and also cell 3. And it does it by producing molecules that Earl Sutherland called first messengers. Today we call them hormones, we call them cytokines, we call them growth factors, we call them a variety of things, paracrine substances, autacoids etc. They come in different shapes and sizes and flavours. Some are small amino acids; some are large proteins like the cardiac troponins. The point is they’re released into the interstitial space in the blood stream and they home the body to find its target. It identifies its target cell by the presence in their membrane of a macromolecule that we call a receptor. Sometimes these receptors are inside of the cell, as with steroids and thyroid hormone, but most often they’re transmembrane proteins, integral membrane proteins in the membrane surface. These ligands or first messengers interact with their appropriate cells that only possess those receptors. That’s where the specificity of the reaction occurs. They fit together conformationally, like a key in a lock. And they then only perturb the cells that have the appropriate receptor. The ligand doesn’t necessarily have to enter inside of the cell to cause the cascade of biochemistry to result in some physiologic response. They can just interact with the receptor, it tweaks it and then all of a sudden, voila, there are lots of different intracellular second messengers that accumulate. The first such second messenger was cyclic AMP. What Sutherland and Rall discovered: This is how glucagon and epinephrine regulated glycogen degradation in the liver. We know today that lots of pathways utilise that messenger. We also know that there are other second messengers besides cyclic AMP: cyclic GMP, calcium, diacylglycerol, nitric oxide. And while there are hundreds and hundreds of first messengers, there are a modest number of intracellular second messengers. Perhaps no more than a dozen at present but there will be more, I’m sure, in the future. These second messengers accumulate and carry out the function within the cell that was brought by this first messenger to the cell surface. What is unique about nitric oxide as an intracellular second messenger is that it’s a gas. It’s a free radical with an unshared electron and a very simple molecule. And because it’s uncharged, the physiologic pH, like all the other messengers, it doesn’t go in and out of membranes... it goes in and out of membranes much more readily than the other messengers that often require energy or transporters to do that. So it not only regulates the biochemistry in the cell in which it’s made, nitric oxide, but it can come out and travel a couple of hundred Angstrom, microns, to regulate adjacent cells to produce other second messengers such as cyclic GMP. So it’s a very unique messenger molecule. It’s the only messenger I’m aware of that functions both intracellularly as well as extracellularly. It can be a paracrine substance, a local autacoid. It can also bind with other carriers, glutathione, thiols, albumen, other proteins and be transported a distance to be released again and function therefore as a hormone. No other messengers can do that. Nitric oxide is a very old molecule and I have a theory that it participated also in evolution. It was probably one of the first messenger molecules 3 billion years ago, as cells began to communicate with each other. But I won’t get into that story because time doesn’t permit. But today we know that it’s very important as a pollutant. And this is when it became popular about 50 or 60 years ago when it was apparent that all fossil fuels when combusted with oxygen produced a family of nitrogen oxides, NO, NO2, N2O3 etc. All of these nitrogen oxides will interact with ozone and deplete the ozone layer and are responsible for global warming along with the greenhouse gases. But I’m going to tell you that not only is the nitric oxide a pollutant but it’s the mechanism of action of some very important cardiovascular drugs and it’s a very important messenger molecule in the body. And this is just a partial list of the biology that it regulates. And the list is much, much longer than this. It includes muscle relaxation, platelet aggregation, penile erection, killing of parasites and microorganisms and the list goes on and on. We’ll come back to some of that shortly. In 1963, 7 years after the discovery or 6 years after the discovery of cyclic AMP, a couple of chemists discovered for the first time cyclic GMP, another cyclic nucleotide, a cousin of cyclic AMP. All you do is shift the amino group on the purine ring and you have a different structure. They administered inorganic P32 phosphate to rats, harvested the urine and found two major organic phosphates in urine, cyclic AMP, the other being cyclic GMP. That was the first demonstration of cyclic GMP as a natural product. That stimulated a few laboratories to become interested in cyclic GMP, to look for enzymes that made it, enzymes that hydrolysed it etc. And the story began to evolve in the late 1960s, early 1970s just as I was finishing my training and getting ready to join the faculty at the University of Virginia. We know today that there is a family of enzymes called guanylyl cyclases and there are at least seven or eight members of this family and we think there will be even more with splice variants that we’re now isolating. All of these enzymes convert GTP to cyclic GMP and pyrophosphate. The reaction is very analogous to add an OH cyclase that converts ATP to cyclic AMP and pyrophosphate. In fact the catalytic domains of all of the cyclases are homologous with the change of one or two amino acids, one enzyme can make the other nucleotide. The cyclic GMP can be inactivated by a family of phosphodiesterases; there are at least 10 or 11 members in this gene family. And they hydrolyse the phosphodiester bond to convert the cyclic nucleotide to the corresponding monophosphate, either 5’ GMP or 5’ AMP and it becomes inactive. As you will hear later in the week from Doctor Fischer, many of the intracellular second messengers, cyclic AMP, cyclic GMP, diacylglycerol, calcium etc., often activate a protein kinase that then phosphorylates a variety of protein substrates by transferring the gamma phosphate from ATP to a serine-threonine residue or perhaps a tyrosine residue. There are I’m told now as many as 500 or 600 protein kinases. So while the concept of cell communication is pretty simple, a ligand or hormone regulates the second messenger production which then regulates the protein kinase which phosphorylates a protein, the problem is the matrix gets pretty hairy when you consider all the cyclases, the diastasis, the protein kinases and the numerous proteins that can be phosphorylated. When these proteins are phosphorylated, as you’ll hear from Doctor Fischer, their conformation changes. If they’re structural proteins they can influence motility and other processes, contractility. If they’re enzymes they can be activated or inhibited. So as this story was beginning to unfold with cyclic GMP, I decided to desert cyclic AMP and switch my interest to cyclic GMP. As a new young faculty member in the early 1970’s the cyclic AMP field in my opinion was becoming rather crowded, large groups of people around the world. And I didn’t think that I could compete with all these huge laboratories and cyclic GMP was evolving and I said I’m going to go in this new direction. And I wanted to address two questions. We knew that a couple of hormones could increase cyclic GMP accumulation in a couple of tissues, acetylcholine in heart preparations, prostaglandins in vascular preparations. But we didn’t know the molecular coupling mechanism. If we understood ligand binding to the receptor and the coupling to the guanylyl cyclase to make cyclic GMP, we could presumably influence that coupling process with various chemicals and drugs to potentiate a hormone response or block it and maybe come up with some novel therapies for various endocrine diseases. That was quite rational. The second is, although cyclic GMP was a natural product, we had no idea what it did or what its function was. So we began our studies by looking at the enzyme in some detail that made cyclic GMP and the first big surprise was that there wasn’t just a single enzyme but a couple of enzymes. The enzyme activity was in soluble fractions as well as particulate fractions. The kinetic properties of this isoforms was different. I suspected there would be isoforms. I had thought to myself: If there are different compartments and isoforms of guanylyl cyclase, perhaps they’re regulated by different groups of hormones, wouldn’t that be a fun project to sort out. And a lot of that all turned out to be true intuition at the time. Well, to prove that we were dealing with isoforms was going to take a lot of work. We had to purify these soluble particulate activities, clone them, express them, restudy them. We did that, it took us 12 or 15 years of work to do all that. But initially I took a shortcut. I said the cooperativity of the particulate isoform, whereas the typical Michaelis-Menten kinetics of the soluble could be artifactual because these were crude preparations that possessed nucleotidases, phosphatases, phosphodiesterases. So we created a cocktail and added it to our incubations to inhibit all these competing pathways. We made a cocktail pyrophosphate, fluoride, azide, hydroxylamine, sodium nitrite, methylxanthines etc. And quite accidentally, as science often is the case, we found compounds that activated the enzyme. While our goal was to figure out hormonal regulation, we couldn’t do that because once we disrupted tissues, the hormone coupling was lost. Hormones no longer activated extracts of the enzyme. But the small molecules now would activate and maybe they could be our surrogates for understanding hormonal regulation. Much as fluoride was a valuable resource for understanding hormonal regulation of adenylate cyclase. The activators were azide, hydroxylamine and sodium nitrite. And to make a long story short, the effects of the azide were oxygen dependent, enhanced with thiols, had a time lag before the rate of the reaction became maximal. They were tissue specific because tissues possessed inhibitors and activators of the pathway. And we were convinced that these activators were precursors or prodrugs being converted to something else in our incubation. And it became quite a mystery story for several years to figure out what that activator was going to be. We put azide and hydroxylamine and nitrite on cell cultures in tissues. And indeed they would elevate cyclic GMP levels and would also activate the enzyme and extracts. So they would work in both systems. One of the tissues, again fortunately, that we were working with was tracheal smooth muscle. As a student I knew that cyclic AMP relaxed smooth muscle, vascular and airway smooth muscle. And I thought cyclic GMP might antagonise cyclic AMP. So we prepared a smooth muscle preparation that was relatively homogenous in order to do some biochemistry and compare it with a physiologer. We put azide on those smooth muscle preparations, the cyclic GMP levels went up, but the muscle didn’t contract, it relaxed. The opposite of what we expected. The dose response curves, the time courses, everything told us that azide was a smooth muscle relaxant, mediating its effects through cyclic GMP and that’s how it worked. And that was the first physiologic function of cyclic GMP. We said: If these drugs cause relaxation and elevate G, what do the other smooth muscle relaxants do? Nitro-glycerine, which had been around for 100 years, nitroprusside? A lot of popular drugs in the cardiovascular intensive care units. They decreased after load, lowered blood pressure in patients with infarction and so forth. We put them on our preparations, they relaxed as expected. The surprise was that they elevated cyclic GMP levels. So now we had a family of compounds that we called nitrovasodilators that were capable of activating guanine cyclase in cell free preparations and elevating cyclic GMP levels in various tissues. These compounds included hydroxylamine, azide, sodium nitrite, some hydrazines, nitro-glycerine, nitroprusside, nitrosoureas, nitrosamines and a long list of other compounds, all NO donors. They all have nitrogen. Some are converted spontaneously based on redox and pH incubations. Some are converted insomatically, azide requires catalase conversion. And we reasoned that the intermediate for all of these prodrugs or precursors had to be nitric oxide. And the reason we thought it was nitric oxide is because azide’s tissue specificity was in part influenced by the presence of haemoglobin and myoglobin in our tissue extracts. These were inhibitors. We knew from the literature that NO had a very high affinity for the heme prosthetic group of haemoglobin, myoglobin. So we reasoned that all of these prodrugs or precursors were converted to nitric oxide. When we generated nitric oxide in the fume hood chemically, sodium nitrite, ferrous sulphate, sulphuric acid, ventilated the gas into our incubations, every preparation was activated. What an exciting period that was. The first demonstration that a free radical could activate an enzyme. And I thought that would be interesting chemistry. Lots of folks were sceptical. They all thought of free radicals and the nitric oxides as pollutants and toxic materials and sure enough it activated. Some people argued: Murad, you’re inhibiting an inhibitor, you’re disinhibiting in your crude preparations. So we were obliged to purify the enzyme to homogeneity and when we did the concentration the nitric oxide required to activate kept becoming lower, lower, lower as we moved scavengers and sinks from our incubation. Transition metals, thiols, proteins and sucked it up, you know. Nanomolar concentrations of NO would activate the enzyme and the Vmax would increase 200- to 400-fold, as it would lower the Km for GTP. So we realised that we had figured out the mechanism of action of the nitrovasodilators, such as nitro-glycerine that had been used clinically for 100 years. As a pharmacologist, when you find an exogenous material that does something in a biological system, you should ask yourself: Is it mimicking a natural pathway that’s already there? And is it working through a similar mechanism? So I proposed in some review articles in the late ‘70s that perhaps the effects of various hormones to increase cyclic GMP levels were because they were increasing the production of nitric oxide from some endogenous precursor, perhaps by altering some redox pathway. We couldn’t prove that hypothesis because the technology was not there to measure nitric oxide in its oxidation products at animals or concentrations back in the 70’s. There were crude colorimetric assays. So to prove this took another seven or eight years of technology development before we and others were able to prove that it turned out to be the case. But we expected that nitric oxide was going to be an intracellular second messenger and that was heresy. And it was enough to show that it activates... a free radical activates an enzyme, now you're saying a free radical is a second messenger and it turned out to be true, we turned out to be right. And basically that’s the reason we went to Stockholm. Shortly after this Robert Furchgott, a vascular pharmacologist in New York, showed for the first time that a group of agents would relax vascular segments in the organ bath in the laboratory. Agents such as acetylcholine, histamine, bradykinin that were known to be hypotensive in man or animals had always failed to cause relaxation in the laboratory. He found, if he preserved the integrity of the endothelium in his blood vessel preparations, they would cause relaxation. And that was an exciting turn of events in 1980. I heard him present this work, he said that these substances caused the release of a factor from the endothelium which he called endothelium-derived relaxant factor, or EDRF, and had a half-life of only several seconds. I said: Bob, this is a reactive species, maybe a free radical, maybe it works in cyclic G, maybe it’s going to be related somehow to NO. Let’s figure this out. Well, we moved to Stanford, Bob went to New York, his wife developed breast cancer and that collaboration never took place. But a couple of years later we became impatient and moved on ourselves and showed that that hypothesis was in fact the case. This is a blood vessel precontracted with norepinephrine. And then after five or seven minutes we introduced an endothelial dependent vasodilator, acetylcholine, bradykinin, ATP ionophore, histamine etc. And if the endothelium is present, cyclic GMP levels increased within seconds, returned to basals and this is followed by relaxation in the muscle. But that only occurs if the endothelium is intact, if there’s no endothelium there’s no increase in cyclic G, no relaxation. So we knew by the early 1980s there were now two pathways converging on guanylyl cyclase to regulate cyclic GMP synthesis. The NO donors in the endothelial dependent vasodilator pathway. We spent a couple of years showing that this resulted in the activation of protein kinase G in vascular smooth muscle preparations. We then chased the phosphorylation of a variety of proteins with P32 incorporation. And to put all of the story together for you, it’s summarised on this slide. This cartoon represents a blood vessel with the endothelial lining on the left and the smooth muscle compartment on the right and in red are three categories of vasodilators that work by enhancing cyclic GMP production. The nitrovasodilators that are converted spontaneously based on pH oxygen tension etc. or the enzymatically converted, such as nitro-glycerine, to nitric oxide which activates the soluble guanylyl cyclase. Soluble guanylyl cyclase is a heterodimer with an alpha-beta-subunit and the beta-subunit has a heme prosthetic group at the histamine 105 position. The iron in that heme has to be ferrous, NO binds to the ferrous, changes the liganding of the heme to the beta-subunit which then talks to the catalytic domain down at the C-terminal, resulting in activation of the enzyme. The cyclic GMP activates a cyclic G dependent protein kinase. There are a couple of these, soluble and particulate that phosphorylate numerous proteins. These proteins regulate the concentration of cytosol calcium by altering distribution within the cell from stores or through channels in the membrane. And we know that the myosin light-chain kinase is a calcium calmodulin dependent enzyme. So when you decrease calcium, you decrease the kinase, myosin light-chain accumulates in the dephosphorylated state. If you take actin and myosin filaments, when you phosphorylate the myosin light-chain, they slide together for contraction. You dephosphorylate they slide apart and you get relaxation. Since this work it’s been shown that cyclic GMP also regulates the phosphatase activity perhaps. The endothelial dependent vasodilators do exactly the same thing; however, the receptors for these endothelial dependent vasodilators are only on the endothelial cells, not the smooth muscle cells. That’s why patients with arthrosclerosis and cardiovascular disease get direct acting NO donors, nitro-glycerine, nitroprusside etc. They don’t get endothelial dependent vasodilators because their endothelium is abnormal. And I’ll tell you more about that in a moment. But they produce EDRF, which we know is nitric oxide, to work through the same pathway. A third category are the atriopeptins. It was found some years ago by deBold that the granules and atria possess a peptide called atrial natriuretic factor and we found that that factor, atriopeptins and the family of these, activate the particulate isoforms of guanylyl cyclase and there are several of these. It turns out the particulate cyclases are receptor cyclases. For atriopeptins, guanylyl, e-coli enterotoxin etc. And by elevating cyclic G, they too will cause relaxation. Now, you can relax smooth muscle in other ways with agents that alter calcium, agents that increase cyclic AMP, phosphodiesterase inhibitors etc. These are the mechanisms in which you cause relaxation by enhancing cyclic GMP production. But you can start combining this information now to create novel combination therapies for various cardiovascular diseases and I’ll show you how to do that in a moment. There was some other very important observations along the way and I’m going to have to skip them. But it became apparent by the mid and late 1980s that there had to be a pathway that was making nitric oxide. People were finding nitride and nitrate in their condition media, people were finding arginine activation of the pathway. And it turns out indeed there’s a family of enzymes called nitric oxide synthases, very ubiquitous, found in most tissues. And there are 3 isoforms, initially called neuronal NOS, inducible NOS, endothelial NOS, the tissues which they were first purified and characterised. Because they’re so ubiquitous, I’ve suggested we call them NOS-1, 2 and 3 in the chronological order in which they were first characterised, purified, cloned. Very homologous, about 50% to 60% homology with each other. Some are soluble, some are particulates, lots of opportunity for post-translational modifications. Some are acylated with polmitate or myristate; many of them are phosphorylated by various kinases. A very complicated family. Normally this NOS-2 or inducible NOS is not found in tissues. You don’t see transcript or protein unless there’s been an inflammatory insult. So it’s become a biomarker of inflammation. You can enhance its production by anything that turns on the NF-kappaB pathway, endotoxin, pro-inflammatory cytokines, IO1, interfering gamma etc. And you make a lot of enzyme which is a high output production stage for nitric oxide that will participate in a lot of cytoxic and inflammatory processes. All of these enzymes catalyse this reaction. They oxidise the guanidino nitrogen of arginine. They oxidise the turtle guanidino nitrogen to an intermediate hydroxy arginine, further oxidised NO and citrulline. The enzymes are only active as homodimers. They have a heme prosthetic group. They require NADPH oxygen flavines, heme as a prosthetic group. Very complicated. If the bioptron is oxidised to the dihydro, it no longer makes NO but the enzyme now makes super-oxide, one of the worse things you can do in nitric oxide biology. So let me put this pathway together for you. We know now that various hormones and ligands will interact with their appropriate receptors to regulate various co-factors for the nitric oxide synthase pathways to convert arginine to nitric oxide and cytroline. This then activates soluble guanylyl cyclase to make cyclic GMP which activates a kinase and the cascade goes on to cause some processor event. We can influence this pathway in a variety of ways now with pharmacological tools. We have compounds that are receptor agonist and antagonist. We can modify the availability of the cofactors. We have compounds that are arginine analogues or guanidino analogues to block and inhibit the nitric oxide synthases. Some of these are in clinical trials for septic shock and inflammatory diseases. We can scavenge the NO not only with haemoglobin and monoglobin but also with various thiols that are also in clinical trials. There are compounds available that activate the cyclase without NO. We have a mutant enzyme that doesn’t require NO for gene therapy some day we think. You can also enhance the activation by NO or another gas, carbon monoxide, which is a weak activator in the presence of compounds, and there are inhibitors available as well. So we have tools now to begin to approach this system. But the system is a little bit more complicated than that, it always is. When you make nitric oxide, what it wants to do is activate guanylyl cyclase to make cyclic G, to inhibit platelet aggregation, to act as a neurotransmitter, to influence smooth muscle relaxation, all the beneficial sorts of things. However it can get oxidised to nitrite nitrate, it can form complexes with other transition metals. It will form complexes with various thiol groups and proteins. More than 100 proteins get nitrosated on the cysteine residues and one of the important pathways is the interaction of nitric oxide with superoxide anion. This reaction is almost diffusion limited. So whenever you’ve got either in contact with one another, that’s the reaction that will take place and it acts as a steal or a sink to pull it away from the cyclic G pathway. And you make peroxynitrite, which is even more toxic than nitric oxide or superoxide. Peroxynitrite will nitrate protein residues adjacent to the hydroxyl and tyrosines, perhaps sterically inhibiting or interfering with tyrosine kinase phosphorylation in some cases. So maybe the two systems are talking to each other. The tyrosine kinase pathway, the NO pathway, perhaps. There’s been very little evidence in vivo showing that, most of the evidence so far as been in vitro. There is an important disease called endothelial dysfunction. Many of us, certainly the older folks in this audience, have endothelial dysfunction, maybe not so many of the younger people yet. It occurs with hypertension, atherosclerosis, diabetes, tobacco use and perhaps obesity. In all of these disorders there’s oxidated stress, the formation of reactive oxygen species, hydrogen peroxide, superoxide anion, hydroxyl radical and the tissue of blood vessels don’t make sufficient quantities of nitric oxide, for a variety of reasons and I’m not going to go through all the reasons. But the net outcome is your blood vessels don’t make enough NO. They tend to be vasoconstricted, you accelerate the atherosclerotic process. You diminish perfusion in diabetic ulcers and lesions. So it’s a vicious cycle, as you get more constricted more reactive oxygen species, less NO. It gets worse and worse. So you’ve got to interrupt it, you’ve got to treat the underlying disease and perhaps come along with some new approaches, perhaps arginine supplementation to make more NO, antioxidants to get rid of the reactive oxygen species. And there are a lot of clinical, a lot of annual studies to support that. There are some clinical studies. The clinical studies have been very controversial. Almost through here, another slide. The nitric oxide field is gone absolutely bananas. Our first paper on the biological effects was in 1977. Almost as many as cyclic AMP, more than G proteins and a lot of other things. It’s incredible. That is about 6,000 to 8,000 papers per year, in the last 5 to 10 year period. That’s 20 to 30 papers a day. There’s no one in the world to keep up with it. It’s very frustrating for all of us in the field. But it’s also very exciting to see this interest and enthusiasm. I’ve created a partial list for you, this slide and the next, to show you where this field is going to go I think in the next decade with regard to a novel drug development and how you can take these biochemical molecular targets that we and others have described and turn them around now in high throughput screens and other assays to find chemical leads to make new therapeutics. We know, as I said, that NO is participating as a neurotransmitter in the brain, in some regions of the brain, both centrally and peripherally. If you make a NOS-1 knockout mouse and induce a cerebral infarct, the size of the infarct is smaller. If you make a NOS-3 knockout mouse, induce an infarct, the infarct is bigger. If you do a double knockout, NOS-1 and NOS-3, the mouse is not very smart anymore, it can’t get through the water maze just to find the flow. So the participations of nitric oxide is transmitters in various regions, also with memory and we certainly haven’t figured all that out. We know it plays a role in fluid production and re-absorption, in the eye for glaucoma, retinal neuronal degeneration. We talked a lot about vasodilation; pulmonary hypertension was mentioned in the movie. It’s been exciting to see that develop. Premature babies maintained in in vivo circulation, they don’t need to profuse their lungs because they get their oxygen from the placenta from their mother. If they’re born prematurely, their blood vessels are constricted, their lungs are not developed, they need surfactant, you need to dilate those blood vessels and they shunt right to left, so they’re hypoxic babies. If you give them a little nitric oxide in the nasal catheter, they dilate the vessels, they don’t shunt quite as much, they become oxygenated. There’s been a marvellous improvement to eliminate the need for ECMO therapy, which is rather nasty. You all know about Viagra. It came out of this research, not us but we developed the concepts. We used to joke in the lab, we’ll fill condoms with vasodilators and NO donors and PD inhibitors, they work. Viagra is an inhibitor of the type 5 phosphodiesterase which is found predominantly in blood vessels, both in the corpus cavernosum as well as the systemic vessels. This is why patients who take Viagra and now take NO donors, nitro-glycerine, can get severe hypertension, arrhythmia, strokes etc. So one has to be careful, it’s not a street drug, it can get you in trouble. It participates in wound healing and angiogenesis, atherosclerosis. If you look at exhalation of NO in your breath, it’s a wonderful marker of the severity of asthma. The more inflammation in your lungs, the more induction of NOS-2, the more NO you make, the more NO in your breath, so the clinician can have you breathe into a bag or tube and if the NO is elevated, he knows that your asthma is out of control and he’ll readjust therapy. It’s been very useful. There are clinical studies for septic shock, platelet aggregation. We know that NO regulates genes, both up and down from micro array studies on cells. We’ve become very interested in stem cell biology, the last several years we know that nitric oxide and cyclic G influence embryonic human and mouse stem cell proliferation and differentiation. We think we can force cells into different lineages by playing games with NO and cyclic G. And there are other applications, this is only a partial list, we can’t do everything. Thank you very much.

Ferid Murad on Nitric Oxide Signalling
(00:27:50 - 00:30:58)


Signalling Beyond Body Borders
After all, chemical signalling does not end at the borders of our bodies. We routinely send and receive chemical signals to and from the environment. Amongst our five senses, the sense of smell and taste are charged with the reception and processing of such signals. Even though the human sense of smell does not belong to the best developed amongst mammals, its power is still impressive. Humans are able to recognize around 10.000 different odours [11]. These may convey a variety of signals – they may warn us of immediate dangers, such as fires, tell us about the quality of food or, as for the so-called social odours, inform us about sex, health, reproductive status or competitive ability of another human [12].

Curiously, the 10.000 odours we can perceive are composed from merely less than 1.000 different small molecule odorants. The number of odours can be so much higher than the number of odorants because odours are characteristic mixtures of several types of odorant molecules. The latter are typically “very small” with a mass of less than 300 Dalton. Molecules of this size are often volatile, an essential prerequisite for perception by the olfactory system.
The question, how exactly the sense of smell is able to differentiate so efficiently between so many odours has long puzzled scientists. Today we know, that the human olfactory system uses a limited range of odorant receptor types to characterize a given odour. Each of these receptors can, to varying degrees, be activated by several odorants. The odorants of a certain characteristic scent thus create a unique combinatorial pattern, which can be recognized and remembered by the brain. The two scientists who unravelled the workings of the olfactory system, Richard Axel and Linda Buck, were honoured with the Nobel Prize in Physiology or Medicine in 2004. Amongst other things they could point out which receptors are activated by which small molecule odorants. They further showed that pheromones, chemical signals directly triggering social responses, are recognized by similar receptors as smells.

In the case of pheromones, the sensitivity of the involved receptors is truly stunning. The first one to point this out was 1939 Nobel Laureate Adolf Butenandt, introduced above as the discoverer of the sex hormones oestrone, androsterone and progesterone. After receiving the Nobel Prize, Butenandt attempted the first-ever isolation of a pheromone, bombykol. It took his lab 20 years to isolate 15 milligrams of bombykol from 500.000 female silk moths. One reason for the slow success was that the female silk moth requires merely tiny amounts of bombykol to attract males. In fact, a couple of molecules suffice to trigger a reaction. Even today, this stunning sensitivity is unmatched by any form of modern analytical instrumentation. At the 1960 Lindau meeting, shortly after the discovery of bombykol, Butenandt told the audience about his experiments with the first pheromone discovered:

Adolf  Butenandt (1960) - From the Biochemistry of the World of the Insects (German presentation)

Ladies and Gentlemen, At earlier conferences here in Lindau, as just mentioned, I spoke several times about problems in the field of insect biochemistry. My decision to choose the same topic to talk to you about today was prompted mainly by a desire to tell you about the progress and achievements that have been made in areas that, for the most part, could only be discussed in earlier lectures in terms of the problems they presented. Unfortunately, in addition to presenting new facts, I cannot avoid covering some ground that may already be familiar to some of you, for which I beg your indulgence. For those of you who have not yet come across these problems, please permit me a preliminary remark. Some of you may question the point of tackling such specific questions as those posed by the insect world. I’d like to counter that opinion with the following observation. You will all be aware that the science of biology has long since abandoned its descriptive efforts and now seeks, with the help of methods borrowed from physics and chemistry, to analyse fundamental life phenomena that apply universally to all organisms. In this way, it is doing its part in pursuing the aim of science to explore the nature of humankind itself and the living world around it. In so doing, it is free to choose its objects and, depending on the question being addressed and the method used, uses a wide range of different organisms in the animal, plant, and microbial realms. And you will also be aware that insects are very often used to solve fundamental biological problems. We should recall, for example, that the laws of classical genetics that apply to all life forms – including humans – were discovered for the most part in insects and that animal behaviour laws were investigated in insects to a large extent. And in particular, the principles discovered in social insects have delighted awestruck researchers over and over again. Also Biochemistry, which aims to analyse the chemical transformations associated with life processes, chooses its objects from among all the organisms on Earth. For over twenty years we at the Max Planck Institute for Biochemistry have been investigating a number of chemical life phenomena that have been made accessible by studying insects. And as so often we have found in pursuing this work that very specific questions that appear meaningful only for the insect world hold the key to explaining generally valid phenomena or yield findings that can be of practical importance to our own social lives. I would like to demonstrate this with the help of several examples. Let’s begin by considering the natural pigments that are characteristic of the insect kingdom. You all know that the numerous pigments that embellish the natural world and delight humans were a major focus of interest for many generations of chemists. Each period, with improvements in analytical methods, introduced new ways to isolate and identify the structure of natural pigments. So for a long time scientists believed that they were well acquainted with all the important and widespread pigments in the natural world, which often served as models for the technical synthesis of organic dyestuffs. Surprisingly, however, they had overlooked the red, brown, yellow and purple pigments of the insect world, including a previously unknown natural pigment type, which we discovered during our research in recent years and which, as we found, figures among the most widely distributed pigments in nature. First, let us see in pictures what these pigments are and where they occur. In the first picture we see a butterfly, the well-known small tortoiseshell, and this butterfly has just shed its pupal case and has secreted what entomologists call pupal secretion. Many butterflies secrete a cleansing liquid like this immediately after pupating. You can see in this case that the secretion, captured here on blotting paper, has a bright red hue. This is a pigment of the red type in the insect world, and the wing markings, insofar as they are red, yellow or brown, also fall into the group of pigments we will be talking about. Wing pigment of our type can also be seen in the next pictures. In the next picture we again see the wings of the small tortoiseshell. Here is the peacock butterfly, here the admiral. All examples of where yellow, red and brown wing pigments occur. In the next picture we see the silver-washed fritillary, such colours, as well as the pretty red markings of this large American moth Cecropia platysamia, belong here. On the epidermis of some insects or larvae are the natural pigments we’re talking about. In the next picture we see the caterpillar of the puss moth, characterised by the fact that it turns deep red immediately before pupating. The pigmentation is just starting here as depicted. Again this red pigment type. In the next picture you see the pretty colour pattern of a grasshopper larva Schistocerca, here we also have the individual pigment patterns, and finally, I would also like to show you the eye of the grasshopper. Many insect eyes are pigmented red, brown or a darker shade. Now, all these pigments that I’ve presented to you, I repeat, as red, brown or even darker shades of pupating secretion, wing pigments, epidermis pigments, and eye pigments in insects are chemically closely related. And based on their universal occurrence in all insect eyes, where they were first discovered, they have been given the collective name ommochrome eye pigments. The occurrence of these substances is not limited to insects. They are also found as eye and skin pigments in all arthropods, for example crabs, as well as in cephalopods and more rarely in other animal classes as well. There is a whole series of ommochromes as chemical entities. As a characteristic and widespread representative of these ommochromes, let us consider the structure of just one substance in the next picture, of so-called xanthommatin. This yellowish brown pigment, xanthommatin, was first obtained in pure crystallised form from the red pupating secretion of around 10,000 small tortoiseshells, which yielded just 100 mg of the pigment. Later, 19 mg was isolated from the eyes of 7,800 blowflies, marking the first time an insect eye pigment was obtained in pure form. Xanthommatin occurs in very many insect eyes. It is one of the pigments found in wings and skin. The structure of this substance was elucidated and definitively confirmed by synthesis. For those of you familiar with the language of chemical formulae, here is the formula of xanthommatin and you can see that it is what is known as a phenoxazone system, that is a tricyclic system with nitrogen, to which a quinoline ring is attached, and here there is also an oxoaminocarbonic acid side chain. This phenoxazone system is found in all ommochromes. We could say that ommochromes are phenoxazone pigments. Typical of xanthommatin and other ommochromes is a characteristic redox behaviour, which is shown here in the picture. This yellowish brown pigment stage is reduced by the addition of hydrogen, resulting in a bright red stage. Here is the structure of hydroxanthommatin with the two attached hydrogen atoms here and the subsequent rearrangement of the double bonds. The red hydroxanthommatin, with the release of hydrogen, is converted back to the yellowish brown product. And this redox behaviour is shown again in the next picture in colour, here the oxidised yellowish brown, here the reduced bright red colour, this behaviour is, firstly, characteristic of the group and, secondly, is unusual for biochemists and dye chemists, because here reduction is accompanied by a deepening of the colour, which is generally not the case. It is interesting, that nature uses both types, the oxidised and the reduced pigment. We know, for example, that the previously mentioned change from the brown colour of the puss moth to the bright red colour before pupation is due to such a reductive process. All ommochromes are phenoxazone pigments, as I said, we regard xanthommatin as the basic substance, which can be converted in various ways into other types of ommochromes. It is interesting to note that the red hydrated pigment stage is not very stable in the presence of oxygen, instead the oxidised pigment is the stable form. What does nature do when it wants to fix the red hydrated pigment stage? It does this by converting xanthommatin into two other pigments of the bright red type, which can be derived from hydrated xanthommatin, and which we have named rhodommatin and ommatin D. In the next picture I would like to suggest how the hydrated red stage is fixed. Here you see again the hydrated form of xanthommatin, but there is only one hydrogen atom here on the nitrogen, down there on the oxygen it is replaced by a residue R. This residue R can be either a sugar residue, in which case it forms a glucoside, or it can be a sulfa residue, a sulphuric acid ester, and whenever such a hydrogen atom is replaced by such groups – through sugar groups, through sulphuric acid – the reduced form cannot be converted as easily, namely only after removal of the residue, into the oxidised form, and in this way nature fixes the hydrated unstable pigments to produce bright red. They are therefore at the same time substances that show the formula types of other ommochromes. Thank you. And the following strikes us as being of general interest. The ommochromes were found to contain a new pigment system which, as I mentioned, had not previously been observed in nature, the phenoxazone system. This system has been known for over 40 years in the field of synthetic dyestuffs, but its much earlier invention by nature has never been copied. Among these phenoxazone-type synthetic dyes, some are used as photosensitisers on photographic plates. Are pigments of this type in the eyes of insects also involved in light absorption, in the visual process, instead of just acting, as assumed, as photoprotection pigments? This is a question for sensory physiologists. The ommochromes of insects were the first phenoxazone pigments to be discovered in nature. Soon after their discovery, other representatives of this pigment type were found in the natural world. Determining the structure of ommochromes provided the key for analysing the pigmentation system of the so-called actinomycins. Antibiotically active metabolic products of ray fungi and also proved to be the key to other representatives of this class in the fungus kingdom. You again see in the top picture the formula of xanthommatin as a class and how this basic substance is converted by substitution in the group of actinomycins, investigated by Brockmann, and in the lower picture you see types of fungal pigments investigated by Gripenberg. According to very recent observations, it is likely that xanthommatin is also a metabolic product of mammals and perhaps even humans. Whether it has any significance in this context is not known. Actinomycins are antibiotics. This means that they are among the modern weapons doctors use against infectious diseases, about which, in the presentation by Herr Domagk yesterday, we heard so many fascinating things. Because the actinomycins include drugs against malignant diseases of the lymphatic system, it is understandable that the ommochromes – initially merely objects of whimsical research – are increasingly attracting the attention of the pharmaceutical industry. The ommochromes however have gained the greatest theoretical importance as traits which are expressed only under the effect of specific genetic factors. That was the topic of my very first lecture in Lindau: “What do we know about the effects of genetic factors?” Entomologists have found that some insect species are unable to produce ommochromes. And genetic analysis of these ommochrome-free species has shown that they differ from ommochrome-containing wild species in that there is a change or mutation in individual genes in the genome. These genes must therefore control the synthesis of ommochromes. This discovery made it possible to analyse the effects of genes for the first time, that is to answer the question of how specific eye traits of an organism are determined by the presence of specific genetic factors located in the cell nucleus. In the next picture I would like to remind you of the classical experiment by Alfred Kühn, who pioneered this development. In this diagram we see the caterpillar and head of the flour moth Ephestia kuehniella, specifically the wild form characterised by dark ommochrome pigmentation. Here is an ommochrome-free mutant, the caterpillar’s skin is colourless, the eyes red, and there is no dark pigment, and these two variants differ only in a single genetic trait. If, as Kühn did, you implant tissue from the wild form into this penultimate instar of the colourless mutant ommochrome, which is otherwise absent, is synthesised in the host under the influence of this tissue, and the colourless insects are transformed into insects that are phenotypically indistinguishable from the wild forms. The implant must transfer a factor that restores the somehow defective process of ommochrome synthesis. The biochemical analysis of this experiment then led to the recognition of a chain of genetic effects, from which the following can be concluded: The ommochromes in our pigments are synthesised from the amino acid tryptophan, a normal product of protein metabolism, by way of stepwise catabolism via kynurenine and hydroxykynurenine. Each of these steps is catalysed by a characteristic enzyme. Those enzymes, however, are present only if certain genes are present in the pool of genetic factors. We therefore conclude the following: the synthesis of the enzymes that catalyse a given reaction step is dependent upon genes. Genes act via enzymes, when genes mutate, the associated enzymes are absent or altered, so that chemical reaction steps, in this case subprocesses of ommochrome synthesis, no longer occur. This experiment showed, for the first time, that genes act via enzymes. Enzymes are the first detectable products of genetic factors. Because enzymes are specific proteins, we also draw the important conclusion from the experimental results that the information required for the synthesis of specific proteins resides in the structure of genetic factors. In this way, a basic problem in biology was discovered and then made accessible by a subsequent experiment. The question as to how genetic factors in a cell convey information for the synthesis of specific proteins and thus for the production of all specific cellular structures is one of the most exciting problems in biochemistry today, the answer to which is probably in the offing. We have insects to thank for teaching us a law that has meanwhile been confirmed on a broad basis in many organisms and that now allows us to analyse control processes in nature that reside primarily in the genetic material. Unfortunately, we cannot continue this thread. We will now turn instead to another control problem in the field of developmental physiology. Most organisms develop from a single cell - the fertilised egg cell – and the question of how this development of the individual is controlled, how each phase of development is causally determined by the preceding phase, defines the complex of problems addressed by developmental physiology. Here, too, insects have taught us many fundamental principles. You will all be aware that the development of many insects proceeds via an interesting metamorphosis. From the egg of a fly or butterfly hatches the larva or caterpillar. As it grows, it passes through stages during which it moults its outer layer, or cuticle. In the process of pupal moulting, the mature larva changes into a pupa. The pupa undergoes further transformation, at the end of which the sexually mature form, the imago, develops. It emerges from the pupa in a process known as imago moulting. It has been found that this sequence of processes, larval moulting, pupal moulting, imago moulting, is triggered and controlled by three specifically acting hormones, and here we are confronted by the second problem, which I’ve already spoken about in Lindau in the past. At the time, it was believed that the three metamorphosis hormones interact according to this scheme, the first hormone, adenotropic hormone, is produced in neurosecretory cells of the brain, under whose effect the prothoracic gland produces a second hormone, prothoracic hormone. Its presence then causes the epidermis, the skin, to moult. The prothoracic hormone triggers moulting. The form of moulting is determined by the presence or absence of the third hormone, which is produced in the corpora allata. When this hormone is present, which is the case during the entire period of larval development, the insect undergoes larval moulting, if the corpora allata ceases to produce this hormone, known as juvenile hormone, pupal moulting occurs, and the same prothoracic hormone is ultimately still required for imago moulting. All three hormones at work here have meanwhile been isolated. And we are able to demonstrate their effects on suitable objects. We will limit our observations today to prothoracic hormone – the actual moulting or pupation hormone. It is the only one of the three to date to have been isolated in crystallised form. From one tonne of fresh silk worm pupae, 75 to 100 mg of the hormone can be obtained, which we have named ecdysone from ecdysis, or moulting. It is a bicyclic system without nitrogen with the formula C18H30O4. Unfortunately, we still do not know the structural details. We now hope to get help from x-ray structural analysis. Nevertheless, in recent years it has been possible, using the pure hormone, to study its physiological effects in detail and in this context I would like to discuss two examples. Here a ligature is being performed on a fly maggot which separates the head end from the rear end. The result of such an intervention, as shown in the next picture, is a partial pupation. Only the anterior end pupates; the posterior end remains a permanent larva. This pattern results because the metamorphosis hormone that induces pupation is produced in the head and cannot travel past the ligature to the abdominal section of the insect. If we inject pure ecdysone into the abdominal section of the permanent larva, it too subsequently undergoes pupation. Here you see how the hormone is injected into the small larva using a microinjection syringe. We inject 0.01 ml of a solution, equivalent to 0.01 grams, or 10^-8 grams of the hormone, to induce pupation. The result of such an injection is statistical in nature, as expected in biological experiments. Here are a number of abdominal sections injected with hormone and you can see that in addition to total pupations, partial pupations have also occurred, while some insects did not respond, as we would expect in a biological experiment. Many of you will recall that this effect forms the basis of the physiological test to detect and isolate ecdysone. The most elegant biological experiment on the effect of ecdysone was conducted by Carrol Williams, and in the next picture, from a paper by Carrol Williams, you can see what he did. Previously we saw the large moth Cecropia platysamia, a beautiful colourful silkmoth, a giant. And here at the top is the isolated abdomen of a pupa of this moth. We see how two pieces of organ are inserted using small needles, namely neurosecretory cells from the brain and prothoracic gland. So the two hormone-secreting glands, under the influence of the other implant, the prothoracic gland, produces prothoracic hormone. This hormone, as I said, also controls the final stage of imago development, and the abdomen of the pupa develops into the abdomen of the moth. The entire morphology changes, the colour changes and the isolated abdomen even lays eggs. It functions. Without the intervention, without a supply of hormone, it would have died. This experiment was repeated with pure crystallised ecdysone instead of the two organ implants. In the next picture we see this isolated abdomen, coloured yellowish brown, placed on a small agar plate, before imago development, before imago moulting. If we did nothing, this isolated abdomen would, of course, die within a short time. After injection of 6 micrograms of ecdysone, the entire developmental process occurs, this is a photo of the same setup. You see the change in the shape of the moth’s abdomen. You see the expression of the colours and the pattern. So the entire developmental process occurs under the influence of a tiny amount of pupating hormone. It would be difficult to demonstrate the causal effect and significance of a hormone in the course of a developmental step more convincingly. Hormones also act as regulators in the developmental stages of mammals, including humans, but nowhere can their effects be studied so easily and in such an isolated setting as here. So how does this hormone actually work? The activity of hormones is, for the most part, not known in detail. We are repeatedly struck by the astonishing fact that such tiny quantities of a substance can trigger such dramatic processes in the organism. I am delighted to tell you that Dr Karlsson, in cooperation with Beermann’s department at the Max Planck Institute for Biology in Tübingen, has recently made an observation with the help of ecdysone which I regard as groundbreaking. For the first time namely it has been shown that the function of gene loci in the nucleus is influenced by this hormone We thank Beermann for demonstrating that those parts of a chromosome in which active gene loci are located undergo a histological change. So-called puffs form, which we interpret biochemically as a loosening of the genetic substance and a transition to its actual function. Geneticists have always claimed that such puffs cannot occur simply from an internal mechanism of the chromosome but that the surrounding milieu must induce the gene loci to carry out specific functions that are required for further development. But it has never been possible to exert an unambiguous influence on puff formation experimentally. Only now has it been possible to demonstrate, using ecdysone and giant chromosomes of dipterans, in which these things can be observed so well, that minute amounts of hormone can induce puffing in highly specific gene loci, thus activating their function. Recalling everything I’ve said before, that in some way the production of specific proteins is initiated from these gene loci, we can imagine that such activation of individual gene loci initiates the production of specific enzymes. That is really just a start. And we also know how we must interpret this effect. And as I said, this is a groundbreaking achievement for which I have the greatest admiration. Ladies and Gentlemen, another example of the control of the development of individual organisms by an active substance is the honey bee, Apis mellifica, in which the expression of various sexual forms will serve as an introduction to the problem of assignment within a social colony. A robust beehive consists of about 50,000 to 80,000 worker bees, 2,000 to 3,000 male bees, the drones, and a single queen. In the next picture we see the morphological differentiation of these types, in the middle the queen, on the left the drones, on the right the workers, you will already be familiar with all this. The drone is nearly as big as the queen but darker. We know that the drones, the male bees, are produced through parthenogenesis. This means from unfertilised eggs. Whereas the queen and workers are produced from fertilised eggs. Whether a fertilised egg gives rise to a worker or a queen is determined solely by how the broods are fed. Here are the various forms of cells, which you are familiar with. A fertilised egg raised in the small cells produces a worker. A fertilised egg raised in a characteristic queen cell becomes a large queen. It has been observed that larvae hatched from the same eggs are fed differently in the various cells. Whereas the worker larvae are fed only a pinhead quantity of worker food, the larva in the large queen cell selected as the queen receives a copious amount of royal jelly, which is produced by glands in the head of nurse bees. In the next picture you see a cross-section through a bee, and here in the head are a number of glands. This one here is the gland that produces royal jelly. Despite much effort - let me reiterate – one gets the impression that whether an egg develops into a queen is determined by royal jelly, that is by the diet. If a bee larva is transferred just after it has hatched in a hive from a normal brood cell, in which it would have developed into a worker, to a queen cell, it is fed royal jelly and becomes a queen. But despite much effort, all attempts were in vain to transform a young larva into a queen outside the hive by feeding it royal jelly. And consequently, even today, many still contend that entirely unknown brood-care factors determine whether a larva becomes a queen. The situation has changed dramatically since Dr Hanser at our institute recently succeeded in identifying the conditions in which just-hatched presumptive worker larvae can be raised in an incubator, outside the hive, into completely normal queen bees solely by feeding them royal jelly, excluding all other brood-care factors. Dr Hanser’s experimental setup is shown in this picture. It is a small wooden board into which, as you can see, small artificial honeycombs are inserted. They consist of small glass vessels, like this one here shown in detail. And you can see here in the vessels the developing larvae floating. They are floating in royal jelly. If one manages to transfer young larvae, within the first two days of hatching, a period during which they are particularly sensitive, living and unharmed to the experimental cells, they will develop in an incubator at a normal hive temperature of 35° and fed with royal jelly, into queens. Larvae, at the end of the second day of life or later, subjected to the same conditions, always develop into workers. However, under the influence of royal jelly, they can develop into giant workers, which rarely occur in normal hives, if at all. The result of the experimental setup is shown here. You see on the left a normal queen from the hive. You see on the far right a normal worker from the hive. This is a queen raised by us in the laboratory, which developed from a small larva that would have produced a worker in the hive. And here is a so-called giant worker, which develops if you introduce the larva to the experimental setup too late, namely when the actual determination has already been made. The queen differs from the workers not only in size, but above all in the development of ovaries and the morphological shape of the mouth parts. Moreover, the queen lacks the tools on her posterior legs required to collect pollen, namely the brushes and baskets, in short: the queen not only differs from the workers in size and the development of ovaries, her entire morphology is different. That’s why it is important to recognize that a queen produced experimentally in an incubator shows all the features of a normal queen, she is even accepted as queen by a colony. In this picture, let me show you the differing morphology of the mouth parts. On the right by the worker with a long proboscis, on the left by the queen. And you can clearly see the morphological differentiation. Now we can therefore draw the following conclusion: It is clear that the decision about the biological fate of a larva is determined in the first 48 hours of its life and indeed solely by the food it is fed. Whether, as we would probably like to assume, royal jelly contains a determinant substance with specific activity or, also a possibility, whether the determination of a larva to develop into a worker is due to poor nutrition during the sensitive period remains uncertain. But the experimental foundation to answer this question has been laid, and we have begun experiments to fractionate royal jelly and test the individual fractions to see if they induce queen development in the incubator. We look forward to next season’s experiments with particular excitement. Ladies and Gentlemen, I would also like to conclude this problem of determination here. The time is already advanced, and in the final part of my lecture I would like to return to a problem that has already been broached here in earlier lectures. The problem regarding communication between insects through chemical substances. We call substances that are produced in an individual, from these transferred to others, and received by those individuals as communication signals pheromones. Pheromones are therefore chemical messengers conveyed from individual to individual. There are many of them and we can regard them as substances of social cohesion, because they are produced in greater numbers in social insects so that individuals can communicate with each other. Pheromones include species- and gender-specific scents produced by insects which are released by the male or female in order to attract a partner and advertise the presence of individuals of the same sex. We have worked with one such gender- and species-specific sexual attractant in the silkmoth. And last year, after 20 years of effort, we were successful for the first time in isolating such a pheromone, such a gender-specific sexual attractant, and determining its chemical structure. In many insects, finding a sexual partner is facilitated by such species-specific attractants. Detailed knowledge about their activity has been gained mainly from butterflies and moths, whose extraordinary olfactory performance was noted very early on. Insect lovers and entomologists have shown through numerous observations that the female of many butterfly and moth species are able to attract mates from afar. I would like to describe an elegant experiment from the recent literature which was carried out on Chinese silkmoths. Males of this moth species were marked individually and released from a moving train at various distances from the home location, at which a female of the species was kept in a gauze cage. From a distance of 4.1 kilometres, 40% of the released males, and from a distance of 11 kilometres 26% returned to the female. By means of a series of easily performed experiments it can be reliably shown that the female in special scent glands produces gender- and species-specific substances which can be extracted from the glands with lipoid solvents and are perceived by the males through sensory fronds on their antennae. We now know that perception of this scent prompts males to fly against the air current, against the direction of the wind, and thus get underway on their quest for a sexual mate. This behaviour explains why males are frequently attracted from large distances. They do not measure for instance the concentration of the molecules, but instead a scent molecule is interpreted to mean “fly off against the direction of the wind”, and we can easily see that in most cases the attractant must have been carried by the wind so that the male’s chances of finding the female are not bad. Many experiments have been carried out to isolate such scents in order to determine their chemical makeup. Usually insect pests such as the nun moth, grapevine moth and gypsy moth are used. And using extracts from the female scent glands of these species, researchers have tried to attract males in the outdoors. The number of males captured served as a measure of the potency of each extract. The knowledge thus gained about the nature of such sexual scents in 40 years of work was extraordinarily poor. In 1939 we undertook the biochemical identification, as I mentioned, of the sexual attractant of the silkmoth Bombyx mori. It had, until then, never been used in such experiments and yet it had the great advantage for the laboratory that it is a domesticated insect which can be easily bred and which is procured in large quantities in the silk industry. The moths no longer consume food, they can be kept in open trays because they do not fly, and in the presence of a female silkworm or under the influence of an extract from the abdominal glands of the female, the practically stationary males become highly excited, beating their wings rapidly and rotating in searching movements. And that forms the basis for the required chemical identification test. We have been, since 1939, in the summer and autumn months, conducting experiments with the aim of identifying this attractant biochemically. For opening up this interesting field, my coworkers and I are indebted to the suggestion and advice of Walter Schoeller, the former head of the main laboratory of Schering AG in Berlin, who, I believe, is also among our friends here today. The choice of the experimental object, this simple experimental object, was made on the advice of the entomologist Görnitz, who, after orientating experiments conducted jointly with Schotte at Schoeller’s Laboratory in Berlin, recognized the possibility of learning about the general character of the sexual attractant of lepidopterans by using the silkmoth as a laboratory model. Here you see a silkmoth and when sexually excited, the female extends two glands from the posteriormost abdominal segment, the lateral sacculi. They are pigmented yellow, and the attractant can be extracted from them with lipoid solvents. The males sit there very calmly after hatching. They can be identified by their large antennae. They maintain their idle state. For the attractant test they can be kept in open trays, where they sit calmly and are used as follows for the test. We first insert a clean glass rod into the glass vessel, position it in front of the males’ antennae and expect no change in the insects’ idle state. When we then dip the tip of the glass rod into an attractant, the insects begin to intensively whirr immediately. For us it is the clearest sign that the insect is still alive at all. These insects live for around eight days without eating, as I mentioned. When you believe that they are outwardly no longer alive, you can determine with this attractant whether they will attempt the very last movements of this kind. You can therefore gauge a reaction to the test in this way. You can define an attractant unit, for example, by saying that one attractant unit of 1 microgram means that the substance, when present in a concentration of 1 microgram per cubic centimetre, elicits this excited dancing in 50% of the experimental insects after we dip the tip of the glass rod, the magic wand, into the solution. We know from the first experiments in 1939 and 1940, which aimed to concentrate and chemically identify the attractant, that the attractant must be a lipoid-soluble, neutral and nonsaponifiable alcohol that is resistant to dilute acids and bases, but sensitive to oxidants. From 7,000 female butterflies we were able at the time to isolate 100 mg of a waxlike fraction that contained one attractant unit in 0.01 micrograms, i.e. 10^-2 micrograms. We believed at the time that we were already pretty close to the goal, but the following years showed that those products would have to be concentrated by several orders of magnitude to obtain the pure attractant. After we, on the basis of many years of gradually improving experimentation, again processed the extract from 500,000 glands the year before last, we finally achieved the goal of purification. As I said, just about 20 years after the work was commenced. We succeeded in obtaining pure attractant in the form of 12 mg from those 500,000 glands and 12 mg of a crystallised coloured ester, from which the colourless attractant itself can be generated. With this small quantity it was possible to carry out an analysis of the chemical structure. The formula of this first gender- and species-specific attractant is given here below. It is a hexadecadienol. That means that we have a straight chain of 16 carbon atoms. Hexadeca is the basic substance. At the end of this chain we have a primary alcohol group-ol, attached to carbon atom 16. We have two double bonds between carbon atoms 4 and 5 and between 6 and 7. Hence, a hexadecadienol. Our picture shows how the essential structural elements were found. The free alcohol was reduced to a known substance, cetyl alcohol, which was unambiguously identified. In this way we showed that a straight chain of 16 carbon atoms was present. The coloured ester was then oxidised using a micromethod specifically developed for this purpose. And all 16 carbon atoms were detected in the form of hydroxycapric acid, in the form of butyric acid and in the form of oxalic acid. Thus, the formula was unequivocally identified. Now, chemists know that the spatial arrangement around a double bond can always give rise to two isomers, resulting from the configuration of the atoms in space. We can have a cis or a trans configuration at the double bonds that carry substituents. This means that a compound with this formula exists in four isomeric forms. A cis can occur at both double bonds, and a trans can occur at both. A cis configuration here, a trans there, or a trans here, a cis there. Spectroscopic analyses have shown that one of the double bonds has a trans configuration, the other probably a cis configuration. Thus, the number of 4 possible isomers has been reduced to 2. In the past year we have attempted to prepare these 4 isomers of hexadecadienol synthetically and have been successful in doing so. These isomers are extraordinarily similar to each other, as you might expect. But the hope of reporting to you today about the physiological testing of the 4 isomers has not been realized, because we still have no silkmoths. This conference was scheduled about 14 days too early. We plan to test these 4 isomers. We are convinced that we have successfully synthesised the attractant itself, and which of the isomers is the actual attractant and how greatly the isomers differ quantitatively in their physiological effect is a question that we are keen to study in the coming weeks. Remarkable is the extremely potent physiological effect of the attractant. The solution used to test the efficacy limit contains, according to the definition of the attractant unit, 10^-10 micrograms per cubic centimetre. The tip of the glass rod, when wetted with the test solution, has around 10^-2 cubic centimetres of test solution adhering to it, corresponding to around 1,000 molecules on the tip. The magic wand carrying this small number of molecules causes 50 of 100 individual silkmoths to beat their wings. When you consider that the vapour pressure of hexadecadienols is not very high and that the vaporised molecules also have to cross the air space between the glass rod and the insects’ antennae, probably very few individual molecules actually reach the sensitive elements of the antennae. Biochemists are astonished by the relatively simple structure of a substance with such specific action, as this substance is perceived as an attractant only by male silkmoths, not by others. And this attractant contains no branches of carbon atoms, no asymmetric carbon atom. Evidently, part of its specificity lies in the configuration of the conjugated double-bond system. Studying the relationship between structure and activity in this example therefore has particularly interesting prospects. Of course, knowledge of the structure of the sexual attractant of the silkmoth is only of theoretical interest. We expect, however, from the outset that the specific sexual attractants of butterflies and moths all belong to the same substance class, so that the Bombyx attractant holds the key to unlocking the secrets of other sexual attractants. This could prove of practical importance in fighting those moths that are feared as plant pests. We know that the insecticides in use today, we will hear more about them tomorrow, are by no means toxic only to insects, they also harm other animal species and plants treated with insecticides can also be detrimental to humans. Unfortunately, along with insect pests, useful insects are also destroyed, and the lack of selectivity in today’s pesticides can lead to major disruptions in the biological equilibrium of the treated biotope. In addition, the use of insecticides leads to the development of resistant strains that are immune to the effects of the agent being used. The notion of using specific sexual attractants to combat insect pests is old and compelling. We could conceivably use a synthetically manufactured sexual attractant of an insect pest to attract only the males out of a district, capture these, and thus interrupt the propagation of the species. The development of resistance to the attractive scent of the coveted females is, I believe, nothing to fear. Ladies and Gentlemen, I’m happy to tell you that a first step in this direction has been made in the chemical field. A working group headed by Haller in Washington, using the methods we developed on the silkmoth, recently isolated the sexual attractant of the gypsy moth, prepared it in pure form, and analysed it. The gypsy moth ranks among those insects that cause untold damage. And here’s the gratifying news for us. What is the chemical nature of this substance? It also contains 16 carbon atoms in a straight chain. It is also a primary alcohol, like our substance. But it contains not two double bonds but just one, and in place of the other double bond, it contains an oxygen function, an acetylated hydroxyl group. The positions of the double bond and the acetoxy group are still unknown. But what is known shows that our idea was correct, that the silkmoth holds the key to the secrets of attractants of insect pests. With the attractant of the gypsy moth experiments conducted outdoors have been successful insofar as it is now possible, with the help of this substance, to determine in a biotope whether gypsy moths are present or not, which is important. And even that would represent a gain if our hope of mounting a real campaign with the attractant is not realized. Ladies and Gentlemen, I would like to stop here. and Thank you for your attention and that I hope I have been able to show you that old problems, which we talked about in the past, are still exciting, and that progress, though unfortunately slow, is being made.

Adolf Butenandt on Pheromones and Bombykol (in German)
(00:47:03 - 01:03:36)

Conclusion
With the pheromones we conclude our excursion through the world of the small molecules of life. Clearly, the important examples of small molecules discussed here represent merely a glimpse on the vast world of biologically relevant small compounds. Some important groups of small molecules, which are not endogenous in humans, had to be by-passed for the sake of brevity. Those include toxins, medicines and drugs. Nobel Prizes related to such substances were, for example, given in 1945 and 1952 for the discovery of antibiotics penicillin and streptomycin, respectively.

Today, small molecules are rather easy to isolate and characterize, especially in contrast to proteins. In addition, sophisticated synthetic methods in many cases allow for their cost-efficient, large-scale synthesis. Beginning in the second half of the 20th century, Nobel Prizes given for the discovery of “new” small molecules have thus become rather scarce. Instead, the elucidation of entire biomolecular mechanisms, often involving a set of already known small molecules, has moved into focus. The 2004 Nobel Prize to Richard Axel and Linda Buck is a good example of that.

Still, it can be stated without doubt that we do not know all biologically relevant small molecules. The future might thus well hold some exciting surprises. And, coming back to the initial theme, small molecules do significantly contribute to the “chemical worth” of a body. As an average human, you make around 70 kilogram of adenosine triphosphate (ATP) per day [13]. If you would purify and sell only a tenth of that, it would instantly make you a millionaire.

Footnotes:
[1]http://www.wired.com/magazine/2011/01/ff_redmarkets/
[2]D.S. Wishart et al., Nucleic Acids Research 2013, 41(D1), D801-7.
[3]Carpenter, KJ, The Nobel Prize and the Discovery of Vitamins, http://www.nobelprize.org/nobel_prizes/medicine/articles/carpenter/
[4]http://lpi.oregonstate.edu/infocenter/paulingrec.html
[5]F.X. Pisunyer, Critical Reviews in Food Science and Nutrition 33 (1993) 359.
[6]Loewi, O. (1960-1961) Perspect. Biol. Med. 4, 3-25
[7]D.M. Quinn, Chemical Reviews 87 (1987) 955.
[8]T.L. Rosenberry, Advances in Enzymology and Related Areas of Molecular
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[9]C. Bremus et al., Journal of Biotechnology 124 (2006) 196.
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