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Luis Federico Leloir, or how to do good science in a hostile environment

2012; Wiley; Volume: 64; Issue: 6 Linguagem: Inglês

10.1002/iub.1031

1521-6551

Armando J. Parodi,

Medical research and treatments

Luis Federico Leloir was born in one of Paris' most upscale neighborhoods (81, Avenue Victor Hugo, a few blocks away from the Arc de Triomphe) on September 6, 1906. According to his own account, his ancestors immigrated to Argentina during the colonial period, coming mostly from southwestern France (Oloron-Sainte Marie, in the Béarn region) and northeastern Spain (Basque Country). Argentine land at the time was inexpensive because of the constant threat of attacks by natives, so the newly arrived went on to buy extensive farms. In time, these would become the lands where the early pioneers would grow the grain and raise cattle that would bring the country wealth and prosperity. Such circumstances allowed Leloir an early dedication to research. At that time, it was very difficult (if not impossible) to get a full time position in Argentina. The reasons for his family moving to Paris are quite sad: Leloir's father was terminally ill, and the Leloirs were in France in search for a cure. Unfortunately, Leloir Sr. passed away before his son was born. His mother brought him back to Buenos Aires 2 years later and sent him to private schools before he finally enrolled in the School of Medicine of the University of Buenos Aires (UBA). Leloir eventually got his M.D. degree in 1932 and practiced medicine at the university's Hospital. It was not long before he approached the head of the School of Medicine's Institute of Physiology, Bernardo Houssay, to start working on his doctoral thesis. Houssay had achieved significant results studying the role of the hypophysis gland in sugar metabolism—results that would earn him the Nobel Prize in Physiology or Medicine in 1947, together with Carl Ferdinand and Gerty Theresa Cori. Leloir and Houssay became friends, a relationship that would last until the latter's death in 1971. Leloir's thesis (Adrenal Glands and Carbohydrates) got the Thesis of the Year Award in 1934. Taking notice of his own shortcomings in sciences considered "harder" than Medicine, Leloir went on to take several doctorate-level Chemistry courses at UBA's School of Sciences. Although he never completed the Ph.D. program for Chemistry, Houssay and other colleagues encouraged him to get further training. Leloir would soon move to Cambridge University (U.K.) to deepen his studies in Biochemistry with Sir Frederick Gowland Hopkins, 1929 Nobel Prize for Physiology or Medicine. Leloir returned to the Institute of Physiology in 1937, where for 6 years he would focus on fatty acid oxidation (together with Juan M. Muñoz) and on the causes of malign nephrogenic hypertension (with both Muñoz and Eduardo Braun Menéndez). In the former study, Leloir and Muñoz prepared liver homogenates that were able to oxidate fatty acids. As trivial as this may sound today, it meant a significant step forward at the time (in fact, the now classic Lehninger's Biochemistry manual cites this finding as one of the milestones of this field). On an anecdotic note, homogenates were subjected to centrifugation to perform a subcellular fractioning and obtain information on where inside the cell was oxidation taking place. Temperature had to be kept down to prevent the material from getting damaged, but Leloir and Muñoz found out that the Institute had no refrigerated centrifuges. They then came up with the idea of covering a regular centrifuge with inner tubes from car tires filled with a refrigerating mix consisting of water, ice, and salt. Although this may seem comical, it shows an amazing ingenuity. Earlier studies by other researchers had demonstrated that if the renal artery in dogs was constricted mechanically, the dog would develop permanent hypertension. Houssay suggested Braun Menéndez to study the causes of this phenomenon since one of his (Houssay's) students had died of nephrogenic hypertension at a very early age. Leloir and Braun were eventually able to explain the mechanism by which such hypertension was generated, with Leloir providing a thorough analysis of the biochemical aspects of such process. In 1943, Leloir married Amelia Zuberbühler, a marriage that would produce a daughter (Amelita) and nine grandchildren. On the public sphere, that same year saw Argentina's civilian government brought down by a military coup d'état. Houssay and several other public figures sent a letter to the new authorities demanding respect for the Constitution, and a return to both democracy and "American solidarity"—the latter being an euphemism for Argentina to align itself with the Allied forces of Word War II. The new military government (in which future President Peron had a decisive influence) was sympathetic to the Axis, reason for which it fired all state employees who had signed the aforementioned letter. Houssay, a professor at a public university, was, therefore, forced to leave his beloved Institute of Physiology and move (together with many of his colleagues) to the newly created, privately funded Institute of Experimental Biology and Medicine. As for Leloir, he moved with his wife to the United States to enhance his knowledge of Biochemistry. He would first work with E. Hunter in Carl and Gerti Gori's laboratory (two Czech scientists based in St. Louis, Missouri, more on them later on this essay), and later on with David Green (with whom he had previously worked in Cambridge) in New York City. In 1945, Leloir returned to the Institute of Physiology shortly after Houssay was reinstated in his post. He immediately started gathering a new team for in his own words he "(didn't) like working alone, but rather to interact with others and exchange ideas with colleagues—and more so if they have a sense of humor." Hence, it was just a matter of time until he teamed up with Ranwell Caputto and Raul Trucco. It was mid 1946 and Peron had recently assumed his first constitutional term as president. One of his first measures was forcing Houssay to retire. Although Houssay was only 59 at the time, those responsible for the decision did not seem to care. Ever cautious and wise, Houssay had made sure that the Institute of Biology and Experimental Medicine was not dismantled while working back at the Institute of Physiology. He moved then again to the former institution together with most of his colleagues who had empathized with their mentor. Among them were Caputto, Trucco, and Leloir himself. At this point, Biochemistry professor Carlos Cardini also joined the new group upon being ousted from his post at the University of Tucumán (Northern Argentina) for political reasons. It was at this point when a powerful textile industrialist, Jaime Campomar, contacted Houssay offering him funds to create a new institute for Biochemical Research (although never confirmed, there have always been well-founded rumors that it was actually Cardini that persuaded Campomar for his decision, for they were brothers-in-law). The offered sum was 100,000 pesos a year—a pretty generous sum for the time (equivalent to 25,000 dollars in 1946). Houssay accepted the offer and suggested Leloir as possible director of the new institution, which would eventually become the Fundación Campomar Institute of Biochemical Research (now called Leloir Institute Foundation). The institute was formally inaugurated in November 1947 in a small house in the Buenos Aires Palermo neighborhood, right next to Houssay's own institute. The latter was accessible through Campomar's back entrance, a fact which made it easier for the groups from both institutes to exchange equipment and ideas. Yet, another newcomer at this stage was Alejandro Paladini, the first Campomar Foundation's Fellowship Awardee. Figure 1 shows the Institute's very first team (L to R): Caputto, Paladini, Cardini, Trucco, and Leloir (the latter opening a fridge that is still in use today). Caputto and Paladini can be seen using a home-made Warburg device to measure oxygen consumption. Ironically enough, the device was nicknamed "Nahuel" to satyrize the fashionable nationalistic feeling of the time (the device was named after Nahuel Huapí, a great lake in the Argentine Patagonia). Above: the Campomar Foundation's first research group, circa 1947–48. From left to right: Ranwell Caputto, Alejandro Paladini, Carlos Cardini, Raúl Trucco, and Luis F. Leloir. Because the results from experiments on fatty acid oxidation in bacteria were difficult to interpret, the team shifted the focus of its research. Caputto recalled that while working on his doctoral thesis at the University of Córdoba (Argentina), he had managed to synthesize lactose (the sugar in milk, composed of galactose and glucose) by incubating extractions of mammary gland and glycogen. The team then tried to repeat those experiments, but the results were still far from conclusive. Many years later Leloir would link Caputto's unsound results to the analytical methods employed, which at the time were quite primitive. What Caputto had most likely observed was the formation of another disaccharide (maltose or glucose-glucose) which today we know can be formed by glycogen amylolytic degradation. Leloir suggested then to study not the formation but the degradation of lactose, by then still an unknown biochemical process. As biological system, they chose a yeast (Saccharomyces fragilis) capable of growing in lactose as a carbon source. This yeast is a cousin of the well known as Saccharomyces cerevisiae, a most noble microorganism as it is used for making bread, beer, and wine (see Fig. 2). Cartoon drawn by Leloir depicting the low spirits of the group while trying to solve UDP-Glc structure. The drawing depicts a sinking ship with its mast holding a pennant that reads "CoCo" ("CoCo" standing for UDP-Glc, the second cofactor involved in the transformation of glucose to galactose, the first one being glucose 1-6 diphosphate), with "PA" (Paladini) trying to save himself and Leloir almost totally drowned. In 10 years' time, Leloir's team was able to describe the general mechanism for the formation of di-, oligo-, and polysaccharides. In these transformations, the so-called nucleotide-sugars play a key role, the first of which to be characterized by Leloir and his group being Uridine Diphosphate Glucose (UDP-Glc). Determining the UDP-Glc structure was a real "tour de force" for the group given the precarious equipment and available techniques as well as the little existing knowledge on the properties of compounds that are totally familiar to us today. They first determined that UDP-Glc had a glucose residue and two phosphates, but the compound had an absorption spectrum at the ultraviolet light that was slightly different from that of compounds containing Adenine, the only nucleotide spectrum known at the time. One day Caputto entered into the laboratory bursting with pride while holding the latest issue of the Journal of Biological Chemistry. Inside it was a description of the spectrum of Uridine, which was identical to that of the compound isolated by Leloir and his collaborators. This is what finally allowed them to complete the UDP-Glc structure. The team's disillusionment while trying to describe UDP-Glc structure is well reflected in a drawing by Leloir from March 8, 1949. The drawing depicts a sinking ship with its mast holding a pennant that reads "CoCo" ("CoCo" standing for UDP-Glc, the second cofactor involved in the transformation of glucose into galactose, the first one being glucose 1-6 diphosphate), with "PA" (Paladini) trying to save himself and Leloir almost totally drowned. Leloir at the bench. UDP-Glc was the first nucleotide-sugar to be discovered, while its transformation in uridine diphosphate galactose (UDP-Gal) indicated nucleotide-sugars' first function, that is, the transformation of one sugar (glucose) into another one (galactose). The team immediately discovered other nucleotide-sugars such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and guanosine diphosphate mannose (GDP-Man). The total number of sugar nucleotide-sugars that have been isolated from diverse sources (yeasts, plants, bacteria, protozoa, mammalian cells, etc.) is to this day hard to determine accurately, although rough estimates count more than one hundred. It is impossible to name GDP-Man without mentioning Leloir's collaborator Enrico Cabib, the Institute's third Campomar Fellowship Awardee. Unfortunately Argentina's university autonomy was trampled on after a military coup in 1966 and Enrico left the country shortly after. He went on to pursue a brilliant career in the United States, which continues to this day. It was precisely Cabib who (together with Leloir) described nucleotide-sugars second and by far most popular and relevant role as sugar donors in the formation of di-, oligo-, and polysaccharides. Leloir's team also described the pathway by which nucleotide-sugars are formed. The work of Leloir and his collaborators on nucleotide-sugars inspired countless researchers around the world. One of them, Herman Kalckar (a Dane residing in the U.S. and a very close friend of Leloir) once wittily remarked that he had just circumscribed himself to work in "Place P-Gal" along the "rue Docteur Leloir" (in Biochemistry P-Gal stands for galactose-phosphate, a key compound in the so-called "Leloir pathway" by which galactose is turned into glucose). As we know, glycogen is a polymer formed by the bonding of many glucoses that are vital for metabolism in live cells, from bacteria to mammals. It is synthesized in times of nutritional bonanza and degraded when external energetic supply decreases. This indicates that both formation and degradation of glycogen must be strictly regulated. In the late 1930s, the Coris, husband and wife, proposed a mechanism for the formation of glycogen (in short: glucose — glucose 6-P — glucose 1-P — glycogen), and for this discovery they received the Nobel Prize for Physiology or Medicine in 1947 together with Bernardo Alberto Houssay. However, there were still doubts about the real physiological role of the mechanism proposed by the Coris. It was found, for instance, that physiological conditions that favored the mechanism's activation led to glycogen degradation rather than to its formation. In 1957, Leloir and Cardini reported that actually UDP-Glc was the sugar donor in glycogen biosynthesis. What the mechanism proposed by the Coris actually described was what happens during degradation upon its reversion, and not during the formation of the polymer. Both glycogen formation and its degradation are subjected to a strict regulation so as to respond efficiently to the needs of the organism. It was precisely while studying the activation of degradation while on energetic request by the organism that Edmond Fischer and Edwin Krebs discovered the most universal and relevant mechanism of activation and deactivation of enzymes and other nonenzymatic proteins, that is, the phosphorylation and dephosphorylation of proteins. This discovery earned them the Nobel Prize for Physiology or Medicine in 1992. Furthermore, the study of the hormonal activation in the degradation of glycogen led Earl Sutherland to discover cyclic AMP, the most important of the so-called Second Messengers, a discovery that earned him the Nobel Prize for Physiology or Medicine in 1971. If we add Leloir and the Coris, we see that the study of glycogen formation and degradation resulted in six Nobel Prize awards. Jaime Campomar died in 1956, and the Institute was left with no funding to continue working. As there were no government organizations like those existing today to come to the Institute's aid, Leloir seriously considered closing down the Institute. The situation was eventually solved thanks to a grant from the U.S. Government via the National Institutes of Health (NIH). In 1955, Peron was ousted from power by a revolution, and the government that came from it marked the beginning of a series of positive changes for Argentine science. The year 1958 saw the creation of many institutions dedicated to the promotion of science such as the National Institute of Industrial Technology (INTI), the one for Agricultural Technology (INTA), and the National Council for Scientific and Technical Research (CONICET), the latter inspired by Houssay, who was to be its president and driving force for many years. It was also during this period that Argentine universities regained their autonomy, a fact which allowed UBA's Science School to create its own Institute for Biochemical Research in 1958. This institute was located within the Campomar Foundation Institute for Biochemical Research itself and offered courses for graduate students and laboratories to perform Ph.D. Thesis. Together with the Buenos Aires Institute for Biochemical Research (created in 1983 and financed by CONICET), there were then three institutions that were working together, all sharing the same researchers, equipment and work site. This three-tier association was particularly productive in attracting students (graduate and post-doctoral) as well as funds destined for research. In the meanwhile, the Institute's facilities had become rather inadequate. The physical space available for work had turned pretty meager, thus limiting the incorporation of new researchers and equipment. The building itself also had ostensible deficiencies such as the several water leaks that Leloir himself had managed to contain over the years by building a complex network of home-made internal channels to prevent the water from damaging the books and magazines in the library. Upon knowing about this situation, the Public Health minister of the time offered Leloir an empty building in the Belgrano neighborhood which had formerly been a nun's school (Figure 3). Leaving the old building was nevertheless somewhat sad for most who had been working in it: to quote a title from a famous book by Winston Churchill, the years 1947–1957 had been the Institute's "finest hour." Moving out by no means meant separation of Leloir's and Houssay's institutes: Leloir's functioned in the first and second floors of the new building, whereas Houssay's Institute for Medicine and Experimental Biology took the ground floor and the basement. Enlarging the size of the Institute for Biochemical Research allowed the incorporation of new researchers, the formation of research groups independent from that directed by Leloir, and the eventual research by several groups on fields different from sugar biochemistry. Some researchers in the Institute opposed this subject diversification, but Leloir was not one of them. He firmly encouraged it as a way to enrich research being undertaken. During his first years in the new building, Leloir focused his research on the formation of a cousin of glycogen also formed by many units of glucose, commonly known as starch, a reserve polymer in plants and also a good friend of housewives. As a precursor in the synthesis of this compound, Leloir first used UDP-Glc since (as stated above) this compound worked for glycogen. However, the experimental data obtained showed that the compound was a very poor precursor in starch formation. Leloir then figured that maybe UDP-Glc was not the right precursor and proceeded to chemically synthesize different nucleotide-sugars, all of them with glucose as monosaccharide residue. This is how he found out that ADP-Glc (Adenosine Diphosphate Glucose) was a much better precursor than UDP-Glc. Later on and while studying the composition of nucleotide-sugars in sweet corn, Leloir found out that ADP-Glc was a compound of real biological existence and not a laboratory invention. ADP-Glc also turned out to be the precursor of glycogen synthesis in bacteria. Leloir then moved on to study the diverse properties of high-molecular-weight glycogen (sensitivity to heat, mild acid or alkali, aspect under the electronic microscope, etc..) either isolated from rat liver or formed in the test tube according to the biochemical pathways proposed by him and Cardini or by the Coris. His group reported that glycogen synthesized by both of these procedures in the test tube presented several differences in those features mentioned above. Because the properties of the polymer formed by Leloir-Cardini's pathway were similar to those of the natural polymer, this finding constituted a direct proof that the path described at the Institute for Biochemical Research was that actually taking place in nature. The description of this pathway in the years 1968–1978 (approx.) was Leloir's last grand contribution to Biochemistry. Glycoproteins are particularly important in cell sociology because they are mostly inserted in the plasma membrane, with the sugars on the external side of the cells. This makes sugars attached to proteins the first elements to be recognized when a cell makes contact with another cell or with a protein (such as a hormone). In the mid-1960s, Phillips Robbins (M.I.T.) and Jack Strominger (Harvard University) labs announced that sugars attached to polyprenol lipids took part in the synthesis of several polysaccharides of the bacterial cell wall. The lipid-linked sugars acted as intermediaries between the nucleotide-sugars and the final products. Leloir and his collaborators reported the formation in eukaryotic cells of an oligosaccharide formed by fourteen monosaccharides attached to a polyprenol lipid through a pyrophosphate bridge. The oligosaccharide was then transferred "en bloc" to proteins and further processed (meaning that some monosaccharides were removed while others were added) during the proteins' trip through different internal cell membrane structures before reaching the plasma membrane or the outer cell (secreted glycoproteins). Processing may be different not only in cells of different tissues but also in different glycoproteins within the same cell and even in different oligosaccharides that are attached to the same protein within the same cell. This results in a great variety of oligosaccharide structures within glycoproteins. Such diversity explains the key role of oligosaccharides in the specificity of the recognition processes between cells and between cells and macromolecules. On October 29, 1970 it was announced that the Swedish Academy of Science had awarded Leloir the Nobel Prize for Chemistry for his discovery of nucleotide-sugars and their role in sugar metabolism. I remember that the day before the announcement I saw Leloir in a most unusual behavior (in fact I think I had never seen him do it before): he suddenly stopped the experiment he was doing and laid his arms and head on his desk, as if snoozing. When I asked him what was going on he replied that he had gotten confused on the content of the test tubes and that it was, therefore, useless to go on with the experiment. Shortly after I learned that Leloir had gotten the news about the Prize in advance by a Swedish journalist in exchange for an exclusive interview. Leloir then knew that his days of peace and quiet were over. An unexpected and rather comical consequence was the procession of inventors that gathered at the Institute's entrance to show their inventions to Leloir, who kindly met with many of them. Most of those inventions had to do with the "perpetuum mobile," in open contradiction to the second principle of thermodynamics. Others were scientifically more harmless and perhaps more interesting, such as the secret formula of a very popular cola beverage or reading glasses that prevented the sight from getting tired. The latter consisted of a single glass that got adjusted with a series of strings to be able to read with only one eye. When that eye got tired, it was just a matter of untying the strings and tying them again to adjust the glass to the other eye. Besides the expected official congratulations from the government, the successive public administrations did not show much interest in taking measures to better Leloir's (or his colleagues') working conditions, such as improving the Institute infrastructure or getting new equipment for better research. Finally in 1980, the Buenos Aires Major of the time donated a few lots of municipal ground to the Campomar Foundation, which were located in the Parque Centenario area in the Almagro neighborhood. The city Major also managed the raising of funds to build a new building in those lots—the very first of those occupied by the Institute to be built with the explicit purpose of functioning as a center of biochemical research. The costs were covered in approximately equal shares by both private and public donations. The move to the new building officially took place on December 1983, with Leloir fortunately being able to enjoy the new facilities for 4 years. Leloir was an extremely simple person both in terms of his personality and regarding his interaction with others. He treated everybody he talked to without stiff formalisms but with equal respect and courtesy, regardless of social or cultural distinctions. He was a very calm individual. He never showed anger but became upset whenever someone did not behave politely or made a rude remark about a third party. He hated ostentation and today he would be considered "low key." Even rarer for the Argentina of his time, he shared the direction of his Institute with his colleagues and even his doctorate students so as to make them feel they were all an essential part of a great common enterprise. He was also somewhat shy: he found it difficult to speak in public even when discussing scientific topics. He was pretty unskilled as a speaker and he was visibly upset when lecturing—even while on informal conferences in the Institute itself. I believe one of his greatest pleasures was just being in the laboratory and working with his hands, which he did until not long before passing away in December 1987. Getting the Nobel Prize was undoubtedly a great joy for him, but it also brought him a considerable amount of stress. He also had a subtle and exquisite (and at times even dry) sense of humor which he appreciated when finding it in others, making working by his side a real pleasure. He never had a private office, and he always received visitors and took care of bureaucratic matters (the latter when he had no choice) in his lab. The contrast between his personality and that of Houssay was quite deep in many respects. Houssay was a man of action who constantly visited public officers, state ministers, and even presidents to get financing for the institutions for the promotion of science that he himself founded and directed, such as the Argentine Association for the Advancement of Science and CONICET among several others. However, this contrast in personalities did not interfere with their long-standing friendship. Their relationship was based on a mutual respect for each other's scientific achievements as well as for their common vision on how higher education and research should be in a modern country seeking full development. Leloir was extremely austere when managing funds that were given to him. He would often write the drafts of his scientific papers in ripped sheets of paper already printed on one side. He even used a pencil to allow later corrections with an eraser. Such austerity contrasted deeply with his generosity when dealing with his own money: he always donated his professor salary to the Institute and he himself paid for the maintenance of its library. He was very skilled with his hands and on many occasions designed and built small home-made devices necessary for laboratory work. It is quite common in the science community to find personalities that may be considered as "brilliant." These are scientists who immediately after hearing a conference can provide one or many alternative interpretations to the one that has just been proposed and even suggest countless ideas and related experiments. This was not the case of Leloir, though. After commenting on a published paper or discussing the results obtained in the laboratory, he would take a day or two before speaking out his mind. Only then would he give us a guideline in the form of a comment or a question—one that would set us in the right direction or show us that what we thought we had understood wasn't actually so. I believe that this behavior reflected his wish not to intimidate or offend whoever he spoke with. It may have also been that he needed time for his ideas to grow. Everything he touched (fatty acid oxidation, arterial hypertension, the discovery and function of nucleotide-sugars, glycoprotein biosynthetic pathway) he turned to gold. Once he paved the way for a new subject in Biochemistry he would let the others do the rest. Filling in with the details bored him to death and distracted him from what he really had fun with: doing good science. In the Institute we used to joke about what eventually became known as "Leloir's First Law": number of tubes × ideas = constant. This meant that if we had many good ideas the number of test tubes to obtain good results would be small, while if good ideas were rather scarce the amount of test tubes would have to increase so as to obtain a result worth publishing. It was very common to see him performing his experiments using only two tubes: the test and the control one. Not all of us had that gift. I do not intend to go into details regarding Argentine history. It will suffice to say that whoever gets somewhat acquainted with it would be amazed upon knowing that good science was actually done in this country during a period as unstable as the early 1940s through the early 1980s. Revolutions, coups, military dictatorships, illegitimate civil governments, economic ups and downs, all were the order of the day and virtually few of them promoted or supported science. It was only Leloir's deep love for his country and for those he cared for that made him turn down tempting offers to work abroad. It was also his immense intellectual capacity that allowed him to do science with scarce resources. To him, doing science was in essence a way of having fun. His view on how to do research is well illustrated by the words he wrote shortly after being awarded the Nobel Prize: "one thing I have always tried to avoid is working on subjects that have already drawn other researchers' interest. Young scientists tend to become fascinated with subjects that are in fashion and decide to focus their work on them. By the time they become experts those subjects themselves may already be running out of fashion, or what is worse, they may have become the subject of fierce competition. This whole situation reminds me of the times when I played polo in my youth. The older, more experienced players would always advise me not to ride after the ball itself, for once one reached it, it was already too late. The wise thing to do, they kept telling me, was to ride straight to where one thought the ball will end up. There is a slight time difference between both tactics, and in sports strategy is truly learnt only by experience. When dealing with science, I guess the right strategy is to follow the results from experiments rather than those from literature."