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My Bloch Years: 1961–1963 and Beyond
Biochemical and Biophysical Research Communications 292, 1221–1226 (2002) doi:10.1006/bbrc.2001.2007, available online at http://www.idealibrary.com on
My Bloch Years: 1961–1963 and Beyond Armand J. Fulco 1 Department of Biological Chemistry, UCLA School of Medicine and UCLA Molecular Biology Institute, University of California, Los Angeles, P.O. Box 951737, Los Angeles, California 90095-1737
When I graduated from Loyola High School (Los Angeles) in June of 1950 and matriculated at UCLA four months later, I had no conception of what career I wished to pursue. My father, a physician, hoped that I would choose medicine but, although I elected a premedical curriculum, in part to placate him, I thought it unlikely that I would ever become a medical doctor. I was arguably bright enough to practice medicine but I couldn’t stand to be around sick people. I did enjoy the sciences and creative writing but beyond that, my interests were still too diffuse to settle on a major during my first two years of college. Indeed, I was so distraught by my indecisiveness about a future career, my poor grades and continued family pressure to prepare for a career in medicine that I took the easy way out and, during the fall semester of 1952, volunteered for induction into military service during the Korean conflict. Two years in the army, spent mostly as a stevedore aboard ships in Alaskan and Arctic waters or at my home base in Seattle, gave me abundant time to read and think about the future. Before I returned to UCLA in October of 1954, I had not only decided on a major (chemistry) but had also mapped out a detailed career plan that went something like this: I would continue at UCLA while earning a B.S. in chemistry and a Ph.D. in either organic chemistry or biochemistry and then spend two postdoctoral years elsewhere (preferably at Harvard), before returning to my beloved UCLA as a faculty member to devote the rest of my life to research and teaching. Essentially (there were a few minor glitches and adjustments), that’s how my life turned out and I’ve never regretted it. The key to this outcome was provided principally by Konrad Bloch as I shall explain shortly. After graduating with a B.S. in chemistry in June 1957, I chose biochemistry over organic chemistry for my graduate work, chiefly on the cogent advice from two professors of physiological chemistry, Sidney Roberts and Ralph McKee. For three years, I had worked in their research laboratories as a lab helper and lab tech, and the income from these jobs plus the stipend obtained from the U.S. government via the G.I. Bill allowed me to support myself, my wife, Virginia, and, son, William (with a second child, Lisa, on the way). 1
E-mail: [email protected].
Upon graduation, I was accepted for the fall semester into the Department of Physiological Chemistry (now Biological Chemistry) located in the new UCLA School of Medicine but financial constraints limited my choice of potential mentors to those who would be willing to hire me as a research assistant full time during the summer, prior to the official start of classes in late September when G.I. Bill funding kicked in again. The well-funded mentor turned out to be James “Jim” Mead, a renowned lipid biochemist [died, Nov. 1987], who was then pioneering the use of 14C-labeled fatty acids to study the pathways of unsaturated fatty acid biosynthesis and the transformations of the “essential fatty acids” in higher animals. He had already demonstrated the conversion of linoleic to arachidonic acid as well as delineating the pathway for this conversion (the “Mead Pathway”). Although I knew almost nothing about lipids, I was thrilled to be accepted as a graduate student by a leader in that field and I worked enthusiastically and industriously, often 16 hours a day, on my Ph.D. project, “The Biosynthesis of Polyunsaturated Fatty Acids.” My thesis work, completed in about 30 months, resulted in the publication of two research papers in the Journal of Biological Chemistry (1, 2). Our work was well received, demonstrating as it did, the concept of unique polyunsaturated families in higher animals (exogenous 3 from ␣-linolenate and 6 from linoleate as well as endogenous 7 and 9 from palmitoleate and oleate respectively). I was anxious to begin the next phase of my master plan, postdoctoral work. To this end, I asked Jim Mead who he would consider to be the best lipid biochemist in the world; that would be the person with whom I wanted carry out my postdoctoral research for the next two years. He thought awhile and named three: the team of George Popja´c (who later became a professor in biological chemistry at UCLA), and J. W. Cornforth in England, and Konrad Bloch at Harvard. He then thought a bit more and advised that, if he were me and he had to choose one, it would be Konrad Bloch. I wrote letters of inquiry to Popja´c and Bloch (with concurrent, and I assume laudatory, letters of recommendation to each from Jim Mead) and I received positive responses from both. By this time (spring 1960) we had a third child on the way and the prospect of traveling to England and supporting a growing family on a relatively small U.K.
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fellowship was a bit scary; also, although both Konrad Bloch and the Popja´ c/Cornforth team were most widely known for their research on the pathway for cholesterol biosynthesis, Popja´ c continued that focus while Bloch (who impressed me tremendously when I met him in 1960 at a conference held at UC Davis) had now branched out into research on unsaturated fatty acid biosynthesis that encompassed studies on both the anaerobic pathway in E. coli as well as the oxygendependent processes in yeast and higher organisms. While cholesterol biosynthesis was interesting, the major breakthroughs in this field had been made and, to my mind at least, what remained was to study the multitude of individual steps in detail, surely a heroic undertaking, but, for me at least, not as exciting and pioneering as carrying out research on the as yet incompletely delineated pathways and unexplored mechanisms of unsaturated fatty acid biosynthesis. Furthermore, for me, the learning curve wasn’t very steep and microorganisms (I surmised) would be more compliant research subjects than rats or other mammals. Finally, Dr. Bloch 2 sent me an acceptance letter which indicated that, although he would be on sabbatical leave in England during much of the 1960 –1961 academic year, I was welcome to begin postdoctoral research with him in the 1961–1962 academic year with full salary support if needed. This suited me perfectly; it would give me sufficient time to generate my own support by applying for a 2-year USPHS post doctoral fellowship to work with Dr. Bloch (I received the award) and it also provided an “interim” year (June 1960 –July 1961) to pursue research in Jim Mead’s lab on the origin of the extremely long-chain fatty acids of brain sphingolipids, a project that very much interested me and for which I was supported by a NIH postdoctoral training grant. This research resulted in two publications (3, 4) that, together, clearly established the basic pathways for the formation of the common C 24 fatty acids (lignoceric, cerebronic and nervonic) of the brain and central nervous system. 1961–1963 With my interim year completed and my NIH fellowship in hand, I, my wife, our 3 children, and my wife’s orphaned younger sister, who would be living with us, boarded a cross-country flight to Boston and landed at Logan International Airport on a mid-August morning. I can’t say the first day in our new environment was an unalloyed joy. As soon as we deplaned we started to perspire in the 90°F heat and ⬃100% humidity, a for2 In this article, when discussing the period encompassing my tenure in Konrad Bloch’s laboratory at Harvard (August 15, 1961– August 15, 1963), I refer to him as “Dr. Bloch” as I did then. In discussing events beyond that time, I refer to him by his first name, “Konrad,” the appellation he preferred for use by those of us who had progressed beyond the post doc stage.
bidding combination we had never encountered in arid Los Angeles. Wringing wet but fired by the anticipation of new adventures in an exotic land, we secured a one-month lease for a two-bedroom (but nonairconditioned) apartment in Cambridge within a 15minute walk from Harvard Square. My first week at Conant Labs involved tidying up the lab bench to which I had been assigned by John Law, a former Bloch protege´ and now assistant professor who was acting caretaker while Dr. Bloch was on sabbatical leave, and meeting my new lab mates, 3 Bill Lennarz and Anne Norris who kindly (I think . . .) provided me with rules of behavior and a brief (but incisive) profile of each of the graduate students, postdocs, researchers on sabbatical leave and junior faculty that constituted the Bloch research group and fellow travellers. When Dr. Bloch returned the following week, he welcomed me warmly, inquired about the welfare of my family and set up a meeting in his office for the following day to discuss possible research projects. The meeting, which lasted almost an hour, went well, I thought; I not only emerged with my first project but I also felt that I gained some insight into Dr. Bloch’s approach to science. He had listed about 20 projects on the blackboard in his office and as we went through them one by one, he briefly summarized the goal and research plan for each. I could detect his favorites (which were not necessarily mine) by his inflections, by his facial expressions, and by the amount of time he spent on his narration. I expressed interest in four projects, none of which were on his “favorites” list and he responded kindly, even sympathetically but subtly made it clear that I would be swimming alone in deep waters should I select any of those four. At the same time he gently nudged me in the direction he wanted me to take and, before I knew what was happening, I had selected the very project that (as he said immediately after my choice) was the one he most favored! Briefly, it involved experiments on Clostridium butyricum, a strict anaerobe, to determine whether there was a feedback mechanism regulating the biosynthesis, in this species, of unsaturated fatty acids which included cis-9- and cis7-C 16:1 and their chain-elongation products, cis-11- and cis-9-C 18:1 (5, 6). I spent several months on the project but the results I obtained did not encourage me to go on (and Dr. Bloch agreed). Regardless of whether the bac3 My two years in Konrad Bloch’s research group were enriched not only by my contact with Dr. Bloch himself but also by my interactions with a legion of remarkable people who, during all or part of that period, were working in the Bloch suite of laboratories. These included, in alphabetical order, Blanche Alter, Bernard Babior, Marie Bade, Paul Baronowsky, Ray Clayton, Howard Goldfine, Maria Hulenicka, Joe Erwin, Norah Johnston, Andy Kandutsch, Prof. Katsuki, Norm Lasser, John Law, Bill Lennarz, Ephraim Levin, Robley Light, Franz Meyer, Anne Norris Baldwin, Ben Preiss, Fredo Ritter, Janine Rondest, George Schroepfer and John Sims. I apologize to those whose names have slipped my mind and to others whose names I’ve misspelled.
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teria were grown at a given temperature in the presence of added oleate or palmitate or without the addition of either, the relative and absolute amounts of the two C 16 unsaturated fatty acids, cis-9 and cis-7 in a ratio of ⬃1:4, did not significantly change (the C 18 unsaturated fatty acids were not analyzed since, when oleate was added there was no way to distinguish between exogenous and endogenous components). The saturated fatty acids (again, palmitate was excluded from the calculations) did not noticeably change either under the same conditions. Since both oleate and palmitate (labeled with 14C as a tracer) were taken up and incorporated into lipids by C. butyricum, it appeared that there was no feedback mechanism that would respond to exogenous fatty acids taken up from the medium that could regulate the levels of the endogenous fatty acids. I should note, however, that it was later demonstrated in Howard Goldfine’s laboratory that, to alter the fatty acid composition noticeably in C. butyricum in response to exogenous fatty acids, one must prevent endogenous synthesis (7). I returned to the blackboard in Dr. Bloch’s office and this time we were in agreement from the beginning. I wanted to work on O 2-dependent fatty acid desaturation in a bacterial system and, somewhat to my surprise, Dr. Bloch was full of enthusiasm for the project, something I had not perceived for the same proposal two months previously. This time, the goal was to isolate and characterize a cell-free fatty desaturating system from a strict aerobe, Mycobacterium phlei (M. phlei ATCC 356). During the period 1958 –1960, Bloomfield and Bloch showed (8, 9) that, a cell-free, particulate preparation from the yeast Saccharomyces cerevisiae in the presence of O 2 and NADPH or NADH could carry out the desaturation of stearate to oleate. Essentially, we wanted to compare unsaturated fatty acid biosynthesis in M. phlei with that in the yeast system. This work, carried out over a period of about a year, was fun, exciting and professionally rewarding (10, 11) although, by my own choosing, 12-hour days, 6 days/week were the rule rather than the exception. Essentially, this research resulted in the first O 2dependent cell-free desaturation system from a bacterium and established the basic co-factor requirements for this system (O 2, NADPH, FAD or FMN, Fe 2⫹ and the enzyme, partially purified from 100,000g particles (membrane fragments). These were all absolute requirements; for example, Fe 3⫹ did not replace Fe 2⫹, even in presence of all other components of the system. At the same time that the M. phlei work was in progress, Dr. Bloch kindly let me participate independently in a collaboration with colleagues in Japan and Los Angeles on the pathway for the conversion of linolenic acid to C 20 and C 22 polyunsaturated fatty acids in fish (12). Once the M. phlei work was concluded, Dr. Bloch and I returned to the office blackboard and, again with
mutual enthusiasm, decided to compare O 2-dependent desaturation in M. phlei with desaturation in bacterial species in three other distinct genera including Corynebacterium diphtheriae, Micrococcus lysodeikticus, and Bacillus megaterium. The rationale for choosing these organisms was to test the emerging dogma of the time that, among prokaryotes, the O 2-dependent pathway occurred predominantly in the morphologically and physiologically “more advanced” forms as had been reported for phytobacteria (known at that time as bluegreen algae), and in representatives from orders like the Actinomycetales, Beggiatoales, and Myxobacteriales while “more primitive” prokaryotes (e.g., orders such as the Pseudomonadales and Eubacteriales), aerobic or not, would have the anaerobic pathway for unsaturated fatty acid synthesis (as was true for several Pseudomonas species we tested and for E. coli). In our selection, then, C. diphtheriae would be a representative of a “more advanced” form, since it showed close structural and metabolic similarities to the Mycobacteria (i.e., Mycobacterium phlei) while M. lysodeikticus, and B. megaterium (both of which produced predominately branched-chain fatty acids) would fall into the “primitive” category. How could I know at the time that, in large part due to Dr. Bloch’s generosity, my experiments in his laboratory with B. megaterium, would set the course of my research for the rest of my professional life? As we reported in our JBC paper (13), 4 we were surprised to find that not only C. diphtheriae but also M. lysodeikticus, and B. megaterium required molecular oxygen to produce unsaturated fatty acids. Both C. diphtheriae and M. lysodeikticus produced ⌬ 9monounsaturated fatty acids from added labeled palmitate or stearate but B. megaterium biosynthesized the previously unknown ⌬ 5-C 16 and ⌬ 5-C 18 isomers. The most significant discovery, at least for my future career, was the remarkable temperature dependence of the desaturation reaction in B. megaterium. Thus, at a 23° growth temperature, [1- 14C]palmitate added to the medium was almost completely desaturated to ⌬ 5-C 16 while at 30°, desaturation was negligible. Presumably, this inverse relationship between temperature and desaturation functioned in the regulation of membrane fluidity in response to fluctuations in growth temperature but the mechanism was totally unknown. I could not follow this up in Dr. Bloch’s laboratory, however, because my two-year fellowship was almost at an end. I had declined Dr. Bloch’s generous offer of financial support for an additional two years, followed, possibly, by an assistant professorship position at Harvard, because I was now anxious to 4
Because of a typographical error, the title of this paper, as published, contained the nonsense term “⌬-5-Monosaturated” rather than the correct “⌬-5-Monounsaturated” fatty acids. Perhaps as a consequence, this paper was not referenced in Chemical Abstracts nor, to my knowledge, does it appear in any other major data base.
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return home (specifically to Los Angeles and UCLA) to begin my career as an independent scientist. I think he was rather shocked and disappointed when I did not apply for an opening (which he solicited) for an assistant professorship in biochemistry at UC Berkeley (after all, it was in California and at the most renowned UC institution!) but instead I applied for (and received) a non-tenure track research position at the Laboratory of Nuclear Medicine and Radiation Biology at UCLA. Nevertheless, he remained strongly supportive and, when I later applied (1965) for an assistant professorship in the Department of Biological Chemistry in the UCLA School of Medicine, he wrote a strong letter of support on my behalf to Emil Smith, then chair of that department. The fact that he and Emil were friends and that Konrad Bloch had, some months before, been awarded the Nobel Prize in physiology or medicine certainly did not lessen the impact of his recommendation; I got the position and have been in that department since. AND BEYOND Once I’d secured the research position at UCLA (August 1963) I began casting about for a project. Although I really wanted to work on elucidating the mechanism of temperature-mediated regulation of unsaturated fatty acid biosynthesis in B. megaterium, I dared not even consider this undertaking. I was in full agreement with the rather strict protocol of that time holding that a project begun by a postdoc in the mentor’s lab should remain in that lab when the postdoc left. Indeed, I never broached the subject with Dr. Bloch because I felt that even suggesting such a thing would show an insensitivity and a lack of scientific decorum. Instead, I began working on a variety of other projects, including the pathway for O 2-dependent biosynthesis of unsaturated fatty acids in higher green plants (14), the role of ␣-linolenic acid in photosynthesis (15), and the pathways for chain elongation, 2-hydroxylation, and decarboxylation of very long chain fatty acids in yeast (16). Although each of these projects interested me and allowed me to begin publishing independently, I still could not forget the B. megaterium desaturation system and I often wondered whether Konrad’s group had made headway in elucidating the mechanism of temperature-mediated regulation of desaturation. In June of 1965, I, a multitude of other “Blochheads,” and Konrad himself attended the Gordon Research Conference on Lipid Metabolism at Kimball Union Academy in Meriden, NH. For the Blochheads in attendance, it was our first “post-Nobel” reunion and the event was as much a festive holiday as a scientific conference. Nevertheless, science never took a back seat. During a hike in the woods surrounding Meriden with Konrad Bloch (Fig. 1), Bill Lennarz, Howard Goldfine, and John Law, Konrad asked me how my research
FIG. 1. Professor Konrad Bloch at the Gordon Research Conference on Lipid Metabolism at Kimball Union Academy, June 1965, on a hike in the woods surrounding the Academy.
on lipid metabolism in B. megaterium was coming along. I was dumbfounded by his question since I was about to ask him the same thing! I explained that I was not working with B. megaterium since I had assumed that the project would go forward in his lab where it originated. He responded that the project had originated with me and he took it for granted that I would continue to work on it, given my enthusiasm for elucidating the mechanisms involved in the regulation of membrane fluidity as well as other aspects of lipid metabolism in this bacterium. When he saw that I remained enthusiastic, but had been deferring to him, he urged me to take up the project again. I did so with his blessing, and resumed work on the membrane fluidity problem as soon as I returned home from the conference, publishing five “foundation papers” between 1967 and 1970 (17–21). Indeed, research on lipid metabolism in Bacillus megaterium became my breadand-butter and, although we occasionally strayed to work on other projects, between 1967 and 1998 we published 50 research papers as well as 13 books, book
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postdoctoral research.” When he answered “Konrad Bloch” I took his advice and I’ve never looked back with anything but wonder at my good fortune. Thanks, Jim. REFERENCES
FIG. 2. Professor Konrad Bloch at the Gordon Research Conference on Lipid Metabolism at Andover, N.H. June 1967, playing tennis (his opponent, not pictured, was Howard Goldfine).
chapters, and reviews primarily addressing lipid metabolism in B. megaterium. During most of this period Konrad and I kept in touch, occasionally by phone, but most often in person at scientific meetings (Fig. 2), and he never failed to ask me how our research on B. megaterium was coming along. The last time I saw him was in October of 1995 when I stopped by his office at Harvard on my way back from a P450 meeting at Woods Hole. Although by this time his memory was starting to betray him, he continued to show an interest in my research, and even indicated he had read several of our JBC papers on the mechanisms of cytochrome P450 induction by barbiturates in B. megaterium. In closing this brief history, I will not try to retrace or summarize my research 5 during the past 38 years, except to point out that almost all of it was either initiated during my two postdoctoral years with Konrad or else derived indirectly from research undertaken during that period. However, I will allow myself to reminisce once more about how it all began with a seemingly naive question I addressed in 1960 to my Ph.D. advisor, Professor James Mead. I asked him “Who is the best lipid biochemist in the world . . .? That’s the person with whom I want to carry out my 5 For those readers who may be interested, progressive summaries of my research to about 1997 can be found in a number of reviews, books, and book chapters listed in the references section (22–29).
1. Fulco, A. J., and Mead, J. F. (1959) Metabolism of essential fatty acids. III. Origin of 5,8,11-Eicosatrienoic acid in the fat deficient rat. J. Biol. Chem. 234, 1411–1416. 2. Fulco, A. J., and Mead, J. F. (1960) Metabolism of essential fatty acids. IX. The biosynthesis of the octadecadienoic acids of the rat. J. Biol. Chem. 235, 3379 –3384. 3. Fulco, A. J., and Mead, J. F. (1961) The biosynthesis of lignoceric, cerebronic and nervonic acids. J. Biol. Chem. 236, 2416 –2420. 4. Mead, J. F., and Fulco, A. J. (1961) Distribution of label from 1-C 14-acetate in brain stearic acid. Biochim. Biophys. Acta 54, 362–364. 5. Scheuerbrandt, G., Goldfine, H., Baronowsky, P. E., and Bloch, K. (1961) A novel mechanism for the biosynthesis of unsaturated fatty acids. J. Biol. Chem. 236, PC70 –71. 6. Goldfine, H., and Bloch, K. (1961) On the origin of unsaturated fatty acids in Clostridia. J. Biol. Chem. 236, 2596 –2601. 7. Khuller, G. K., and Goldfine, H. (1975) Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids. Biochemistry 14, 3642–3647. 8. Bloomfield, D. K., and Bloch, K. (1958) Biochim. Biophys. Acta 30, 220. 9. Bloomfield, D. K., and Bloch, K. (1960) J. Biol. Chem. 235, 337–345. 10. Fulco, A. J., and Bloch, K. (1962) Cofactor requirements for fatty acid desaturation in Mycobacterium phlei. Biochim. Biophys. Acta 63, 545–546. 11. Fulco, A. J., and Bloch, K. (1964) Cofactor requirements for the formation of ⌬-9-unsaturated fatty acids in Mycobacterium phlei. J. Biol. Chem. 239, 993–997. 12. Kayama, M., Tsuchiya, Y., Nevenzel, J. C., Fulco, A. J., and Mead, J. F. (1963) Incorporation of linolenic-1-C 14 acid into eicosapentaenoic and docosapentaenoic acids in fish. J. Am. Oil Chem. Soc. 239, 449 –502. 13. Fulco, A. J., Levy, R., and Bloch, K. (1964) The biosynthesis of ⌬-9- and ⌬-5-monounsaturated fatty acids by bacteria. J. Biol. Chem. 239, 998 –1003. 2 14. Fulco, A. J. (1965) Biosynthesis of ⌬-9-monounsaturated fatty acids in slices of the ice plant, Carpobrotus chilense. Biochim. Biophys. Acta 106, 211–212. 15. Appleman, D., Fulco, A. J., and Shugarman, P. M. (1966) Correlation of ␣-linolenate to photosynthetic O 2 production in Chlorella. Plant Physiol. 41, 3608 –3613. 16. Fulco, A. J. (1965) Chain elongation, 2-hydroxylation and decarboxylation of long chain fatty acids by yeast. J. Biol. Chem. 242, 3608 –3613. 17. Fulco, A. J. (1967) The effect of temperature on the formation of ⌬-5-unsaturated fatty acids by bacilli. Biochim. Biophys. Acta 144, 701–704. 18. Fulco, A. J. (1969) The biosynthesis of unsaturated fatty acids by bacilli. I. Temperature induction of the desaturation reaction. J. Biol. Chem. 244, 889 – 895. 19. Fulco, A. J. (1969) Bacterial biosynthesis of polyunsaturated fatty acids. Biochim. Biophys. Acta 187, 169 –171. 20. Fulco, A. J. (1970) The biosynthesis of unsaturated fatty acids by bacilli. II. Temperature-dependent biosynthesis of polyunsaturated fatty acids. J. Biol. Chem. 245, 2985–2990. 21. Fulco, A. J. (1970) Temperature-mediated hyperinduction of
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22. 23.
24.
25.
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fatty acid desaturating enzyme. Biochim. Biophys. Acta 218, 558 –560. Fulco, A. J. (1974) Metabolic alterations of fatty acids. Annu. Rev. Biochem. 43, 215–241. Mead, J. F., and Fulco, A. J. (1976) The Unsaturated and Polyunsaturated Fatty Acids in Health and Disease, Thomas, Springfield, IL. Fulco, A. J. (1977) Fatty acid desaturation in microorganisms. In Polyunsaturated Fatty Acids (W. H. Kunau, and R. T. Holman, Eds.), Chap. 2, pp. 19 –36, American Oil Chemists’ Society. Fulco, A. J., and Fujii, D. K. (1980) Adaptive regulation of membrane lipid biosynthesis in bacilli by environmental temperature. In Membrane Fluidity: Biophysical Techniques and Cellu-
26. 27. 28.
29.
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lar Regulation (M. Kates, and A. Kuksis, Eds.), Part II, pp. 77–98, Humana Press, Clifton, NJ. Fulco, A. J. (1983) Fatty acid metabolism in bacteria. Prog. Lipid Res. 22, 133–160. Fulco, A. J. (1984) Regulation and pathways of membrane lipid biosynthesis in bacilli. Biomembranes 12, 303–327. Fulco, A. J. (1991) P450 BM-3 and other inducible bacterial P450 cytochromes: Biochemistry and regulation. Annu. Rev. Pharm. Toxicol. 31, 177–203. Fulco, A. J. (1998) Barbiturate-inducible gene expression. In Toxicant–Receptor Interactions and Modulation of Gene Expression (M. S. Denison, and W. G. Helferich, Eds.), pp. 103–132 (Chap. 5) Taylor and Francis, Philadelphia.
My Bloch Years: 1961–1963 and Beyond Armand J. Fulco 1 Department of Biological Chemistry, UCLA School of Medicine and UCLA Molecular Biology Institute, University of California, Los Angeles, P.O. Box 951737, Los Angeles, California 90095-1737
When I graduated from Loyola High School (Los Angeles) in June of 1950 and matriculated at UCLA four months later, I had no conception of what career I wished to pursue. My father, a physician, hoped that I would choose medicine but, although I elected a premedical curriculum, in part to placate him, I thought it unlikely that I would ever become a medical doctor. I was arguably bright enough to practice medicine but I couldn’t stand to be around sick people. I did enjoy the sciences and creative writing but beyond that, my interests were still too diffuse to settle on a major during my first two years of college. Indeed, I was so distraught by my indecisiveness about a future career, my poor grades and continued family pressure to prepare for a career in medicine that I took the easy way out and, during the fall semester of 1952, volunteered for induction into military service during the Korean conflict. Two years in the army, spent mostly as a stevedore aboard ships in Alaskan and Arctic waters or at my home base in Seattle, gave me abundant time to read and think about the future. Before I returned to UCLA in October of 1954, I had not only decided on a major (chemistry) but had also mapped out a detailed career plan that went something like this: I would continue at UCLA while earning a B.S. in chemistry and a Ph.D. in either organic chemistry or biochemistry and then spend two postdoctoral years elsewhere (preferably at Harvard), before returning to my beloved UCLA as a faculty member to devote the rest of my life to research and teaching. Essentially (there were a few minor glitches and adjustments), that’s how my life turned out and I’ve never regretted it. The key to this outcome was provided principally by Konrad Bloch as I shall explain shortly. After graduating with a B.S. in chemistry in June 1957, I chose biochemistry over organic chemistry for my graduate work, chiefly on the cogent advice from two professors of physiological chemistry, Sidney Roberts and Ralph McKee. For three years, I had worked in their research laboratories as a lab helper and lab tech, and the income from these jobs plus the stipend obtained from the U.S. government via the G.I. Bill allowed me to support myself, my wife, Virginia, and, son, William (with a second child, Lisa, on the way). 1
E-mail: [email protected].
Upon graduation, I was accepted for the fall semester into the Department of Physiological Chemistry (now Biological Chemistry) located in the new UCLA School of Medicine but financial constraints limited my choice of potential mentors to those who would be willing to hire me as a research assistant full time during the summer, prior to the official start of classes in late September when G.I. Bill funding kicked in again. The well-funded mentor turned out to be James “Jim” Mead, a renowned lipid biochemist [died, Nov. 1987], who was then pioneering the use of 14C-labeled fatty acids to study the pathways of unsaturated fatty acid biosynthesis and the transformations of the “essential fatty acids” in higher animals. He had already demonstrated the conversion of linoleic to arachidonic acid as well as delineating the pathway for this conversion (the “Mead Pathway”). Although I knew almost nothing about lipids, I was thrilled to be accepted as a graduate student by a leader in that field and I worked enthusiastically and industriously, often 16 hours a day, on my Ph.D. project, “The Biosynthesis of Polyunsaturated Fatty Acids.” My thesis work, completed in about 30 months, resulted in the publication of two research papers in the Journal of Biological Chemistry (1, 2). Our work was well received, demonstrating as it did, the concept of unique polyunsaturated families in higher animals (exogenous 3 from ␣-linolenate and 6 from linoleate as well as endogenous 7 and 9 from palmitoleate and oleate respectively). I was anxious to begin the next phase of my master plan, postdoctoral work. To this end, I asked Jim Mead who he would consider to be the best lipid biochemist in the world; that would be the person with whom I wanted carry out my postdoctoral research for the next two years. He thought awhile and named three: the team of George Popja´c (who later became a professor in biological chemistry at UCLA), and J. W. Cornforth in England, and Konrad Bloch at Harvard. He then thought a bit more and advised that, if he were me and he had to choose one, it would be Konrad Bloch. I wrote letters of inquiry to Popja´c and Bloch (with concurrent, and I assume laudatory, letters of recommendation to each from Jim Mead) and I received positive responses from both. By this time (spring 1960) we had a third child on the way and the prospect of traveling to England and supporting a growing family on a relatively small U.K.
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fellowship was a bit scary; also, although both Konrad Bloch and the Popja´ c/Cornforth team were most widely known for their research on the pathway for cholesterol biosynthesis, Popja´ c continued that focus while Bloch (who impressed me tremendously when I met him in 1960 at a conference held at UC Davis) had now branched out into research on unsaturated fatty acid biosynthesis that encompassed studies on both the anaerobic pathway in E. coli as well as the oxygendependent processes in yeast and higher organisms. While cholesterol biosynthesis was interesting, the major breakthroughs in this field had been made and, to my mind at least, what remained was to study the multitude of individual steps in detail, surely a heroic undertaking, but, for me at least, not as exciting and pioneering as carrying out research on the as yet incompletely delineated pathways and unexplored mechanisms of unsaturated fatty acid biosynthesis. Furthermore, for me, the learning curve wasn’t very steep and microorganisms (I surmised) would be more compliant research subjects than rats or other mammals. Finally, Dr. Bloch 2 sent me an acceptance letter which indicated that, although he would be on sabbatical leave in England during much of the 1960 –1961 academic year, I was welcome to begin postdoctoral research with him in the 1961–1962 academic year with full salary support if needed. This suited me perfectly; it would give me sufficient time to generate my own support by applying for a 2-year USPHS post doctoral fellowship to work with Dr. Bloch (I received the award) and it also provided an “interim” year (June 1960 –July 1961) to pursue research in Jim Mead’s lab on the origin of the extremely long-chain fatty acids of brain sphingolipids, a project that very much interested me and for which I was supported by a NIH postdoctoral training grant. This research resulted in two publications (3, 4) that, together, clearly established the basic pathways for the formation of the common C 24 fatty acids (lignoceric, cerebronic and nervonic) of the brain and central nervous system. 1961–1963 With my interim year completed and my NIH fellowship in hand, I, my wife, our 3 children, and my wife’s orphaned younger sister, who would be living with us, boarded a cross-country flight to Boston and landed at Logan International Airport on a mid-August morning. I can’t say the first day in our new environment was an unalloyed joy. As soon as we deplaned we started to perspire in the 90°F heat and ⬃100% humidity, a for2 In this article, when discussing the period encompassing my tenure in Konrad Bloch’s laboratory at Harvard (August 15, 1961– August 15, 1963), I refer to him as “Dr. Bloch” as I did then. In discussing events beyond that time, I refer to him by his first name, “Konrad,” the appellation he preferred for use by those of us who had progressed beyond the post doc stage.
bidding combination we had never encountered in arid Los Angeles. Wringing wet but fired by the anticipation of new adventures in an exotic land, we secured a one-month lease for a two-bedroom (but nonairconditioned) apartment in Cambridge within a 15minute walk from Harvard Square. My first week at Conant Labs involved tidying up the lab bench to which I had been assigned by John Law, a former Bloch protege´ and now assistant professor who was acting caretaker while Dr. Bloch was on sabbatical leave, and meeting my new lab mates, 3 Bill Lennarz and Anne Norris who kindly (I think . . .) provided me with rules of behavior and a brief (but incisive) profile of each of the graduate students, postdocs, researchers on sabbatical leave and junior faculty that constituted the Bloch research group and fellow travellers. When Dr. Bloch returned the following week, he welcomed me warmly, inquired about the welfare of my family and set up a meeting in his office for the following day to discuss possible research projects. The meeting, which lasted almost an hour, went well, I thought; I not only emerged with my first project but I also felt that I gained some insight into Dr. Bloch’s approach to science. He had listed about 20 projects on the blackboard in his office and as we went through them one by one, he briefly summarized the goal and research plan for each. I could detect his favorites (which were not necessarily mine) by his inflections, by his facial expressions, and by the amount of time he spent on his narration. I expressed interest in four projects, none of which were on his “favorites” list and he responded kindly, even sympathetically but subtly made it clear that I would be swimming alone in deep waters should I select any of those four. At the same time he gently nudged me in the direction he wanted me to take and, before I knew what was happening, I had selected the very project that (as he said immediately after my choice) was the one he most favored! Briefly, it involved experiments on Clostridium butyricum, a strict anaerobe, to determine whether there was a feedback mechanism regulating the biosynthesis, in this species, of unsaturated fatty acids which included cis-9- and cis7-C 16:1 and their chain-elongation products, cis-11- and cis-9-C 18:1 (5, 6). I spent several months on the project but the results I obtained did not encourage me to go on (and Dr. Bloch agreed). Regardless of whether the bac3 My two years in Konrad Bloch’s research group were enriched not only by my contact with Dr. Bloch himself but also by my interactions with a legion of remarkable people who, during all or part of that period, were working in the Bloch suite of laboratories. These included, in alphabetical order, Blanche Alter, Bernard Babior, Marie Bade, Paul Baronowsky, Ray Clayton, Howard Goldfine, Maria Hulenicka, Joe Erwin, Norah Johnston, Andy Kandutsch, Prof. Katsuki, Norm Lasser, John Law, Bill Lennarz, Ephraim Levin, Robley Light, Franz Meyer, Anne Norris Baldwin, Ben Preiss, Fredo Ritter, Janine Rondest, George Schroepfer and John Sims. I apologize to those whose names have slipped my mind and to others whose names I’ve misspelled.
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teria were grown at a given temperature in the presence of added oleate or palmitate or without the addition of either, the relative and absolute amounts of the two C 16 unsaturated fatty acids, cis-9 and cis-7 in a ratio of ⬃1:4, did not significantly change (the C 18 unsaturated fatty acids were not analyzed since, when oleate was added there was no way to distinguish between exogenous and endogenous components). The saturated fatty acids (again, palmitate was excluded from the calculations) did not noticeably change either under the same conditions. Since both oleate and palmitate (labeled with 14C as a tracer) were taken up and incorporated into lipids by C. butyricum, it appeared that there was no feedback mechanism that would respond to exogenous fatty acids taken up from the medium that could regulate the levels of the endogenous fatty acids. I should note, however, that it was later demonstrated in Howard Goldfine’s laboratory that, to alter the fatty acid composition noticeably in C. butyricum in response to exogenous fatty acids, one must prevent endogenous synthesis (7). I returned to the blackboard in Dr. Bloch’s office and this time we were in agreement from the beginning. I wanted to work on O 2-dependent fatty acid desaturation in a bacterial system and, somewhat to my surprise, Dr. Bloch was full of enthusiasm for the project, something I had not perceived for the same proposal two months previously. This time, the goal was to isolate and characterize a cell-free fatty desaturating system from a strict aerobe, Mycobacterium phlei (M. phlei ATCC 356). During the period 1958 –1960, Bloomfield and Bloch showed (8, 9) that, a cell-free, particulate preparation from the yeast Saccharomyces cerevisiae in the presence of O 2 and NADPH or NADH could carry out the desaturation of stearate to oleate. Essentially, we wanted to compare unsaturated fatty acid biosynthesis in M. phlei with that in the yeast system. This work, carried out over a period of about a year, was fun, exciting and professionally rewarding (10, 11) although, by my own choosing, 12-hour days, 6 days/week were the rule rather than the exception. Essentially, this research resulted in the first O 2dependent cell-free desaturation system from a bacterium and established the basic co-factor requirements for this system (O 2, NADPH, FAD or FMN, Fe 2⫹ and the enzyme, partially purified from 100,000g particles (membrane fragments). These were all absolute requirements; for example, Fe 3⫹ did not replace Fe 2⫹, even in presence of all other components of the system. At the same time that the M. phlei work was in progress, Dr. Bloch kindly let me participate independently in a collaboration with colleagues in Japan and Los Angeles on the pathway for the conversion of linolenic acid to C 20 and C 22 polyunsaturated fatty acids in fish (12). Once the M. phlei work was concluded, Dr. Bloch and I returned to the office blackboard and, again with
mutual enthusiasm, decided to compare O 2-dependent desaturation in M. phlei with desaturation in bacterial species in three other distinct genera including Corynebacterium diphtheriae, Micrococcus lysodeikticus, and Bacillus megaterium. The rationale for choosing these organisms was to test the emerging dogma of the time that, among prokaryotes, the O 2-dependent pathway occurred predominantly in the morphologically and physiologically “more advanced” forms as had been reported for phytobacteria (known at that time as bluegreen algae), and in representatives from orders like the Actinomycetales, Beggiatoales, and Myxobacteriales while “more primitive” prokaryotes (e.g., orders such as the Pseudomonadales and Eubacteriales), aerobic or not, would have the anaerobic pathway for unsaturated fatty acid synthesis (as was true for several Pseudomonas species we tested and for E. coli). In our selection, then, C. diphtheriae would be a representative of a “more advanced” form, since it showed close structural and metabolic similarities to the Mycobacteria (i.e., Mycobacterium phlei) while M. lysodeikticus, and B. megaterium (both of which produced predominately branched-chain fatty acids) would fall into the “primitive” category. How could I know at the time that, in large part due to Dr. Bloch’s generosity, my experiments in his laboratory with B. megaterium, would set the course of my research for the rest of my professional life? As we reported in our JBC paper (13), 4 we were surprised to find that not only C. diphtheriae but also M. lysodeikticus, and B. megaterium required molecular oxygen to produce unsaturated fatty acids. Both C. diphtheriae and M. lysodeikticus produced ⌬ 9monounsaturated fatty acids from added labeled palmitate or stearate but B. megaterium biosynthesized the previously unknown ⌬ 5-C 16 and ⌬ 5-C 18 isomers. The most significant discovery, at least for my future career, was the remarkable temperature dependence of the desaturation reaction in B. megaterium. Thus, at a 23° growth temperature, [1- 14C]palmitate added to the medium was almost completely desaturated to ⌬ 5-C 16 while at 30°, desaturation was negligible. Presumably, this inverse relationship between temperature and desaturation functioned in the regulation of membrane fluidity in response to fluctuations in growth temperature but the mechanism was totally unknown. I could not follow this up in Dr. Bloch’s laboratory, however, because my two-year fellowship was almost at an end. I had declined Dr. Bloch’s generous offer of financial support for an additional two years, followed, possibly, by an assistant professorship position at Harvard, because I was now anxious to 4
Because of a typographical error, the title of this paper, as published, contained the nonsense term “⌬-5-Monosaturated” rather than the correct “⌬-5-Monounsaturated” fatty acids. Perhaps as a consequence, this paper was not referenced in Chemical Abstracts nor, to my knowledge, does it appear in any other major data base.
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return home (specifically to Los Angeles and UCLA) to begin my career as an independent scientist. I think he was rather shocked and disappointed when I did not apply for an opening (which he solicited) for an assistant professorship in biochemistry at UC Berkeley (after all, it was in California and at the most renowned UC institution!) but instead I applied for (and received) a non-tenure track research position at the Laboratory of Nuclear Medicine and Radiation Biology at UCLA. Nevertheless, he remained strongly supportive and, when I later applied (1965) for an assistant professorship in the Department of Biological Chemistry in the UCLA School of Medicine, he wrote a strong letter of support on my behalf to Emil Smith, then chair of that department. The fact that he and Emil were friends and that Konrad Bloch had, some months before, been awarded the Nobel Prize in physiology or medicine certainly did not lessen the impact of his recommendation; I got the position and have been in that department since. AND BEYOND Once I’d secured the research position at UCLA (August 1963) I began casting about for a project. Although I really wanted to work on elucidating the mechanism of temperature-mediated regulation of unsaturated fatty acid biosynthesis in B. megaterium, I dared not even consider this undertaking. I was in full agreement with the rather strict protocol of that time holding that a project begun by a postdoc in the mentor’s lab should remain in that lab when the postdoc left. Indeed, I never broached the subject with Dr. Bloch because I felt that even suggesting such a thing would show an insensitivity and a lack of scientific decorum. Instead, I began working on a variety of other projects, including the pathway for O 2-dependent biosynthesis of unsaturated fatty acids in higher green plants (14), the role of ␣-linolenic acid in photosynthesis (15), and the pathways for chain elongation, 2-hydroxylation, and decarboxylation of very long chain fatty acids in yeast (16). Although each of these projects interested me and allowed me to begin publishing independently, I still could not forget the B. megaterium desaturation system and I often wondered whether Konrad’s group had made headway in elucidating the mechanism of temperature-mediated regulation of desaturation. In June of 1965, I, a multitude of other “Blochheads,” and Konrad himself attended the Gordon Research Conference on Lipid Metabolism at Kimball Union Academy in Meriden, NH. For the Blochheads in attendance, it was our first “post-Nobel” reunion and the event was as much a festive holiday as a scientific conference. Nevertheless, science never took a back seat. During a hike in the woods surrounding Meriden with Konrad Bloch (Fig. 1), Bill Lennarz, Howard Goldfine, and John Law, Konrad asked me how my research
FIG. 1. Professor Konrad Bloch at the Gordon Research Conference on Lipid Metabolism at Kimball Union Academy, June 1965, on a hike in the woods surrounding the Academy.
on lipid metabolism in B. megaterium was coming along. I was dumbfounded by his question since I was about to ask him the same thing! I explained that I was not working with B. megaterium since I had assumed that the project would go forward in his lab where it originated. He responded that the project had originated with me and he took it for granted that I would continue to work on it, given my enthusiasm for elucidating the mechanisms involved in the regulation of membrane fluidity as well as other aspects of lipid metabolism in this bacterium. When he saw that I remained enthusiastic, but had been deferring to him, he urged me to take up the project again. I did so with his blessing, and resumed work on the membrane fluidity problem as soon as I returned home from the conference, publishing five “foundation papers” between 1967 and 1970 (17–21). Indeed, research on lipid metabolism in Bacillus megaterium became my breadand-butter and, although we occasionally strayed to work on other projects, between 1967 and 1998 we published 50 research papers as well as 13 books, book
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postdoctoral research.” When he answered “Konrad Bloch” I took his advice and I’ve never looked back with anything but wonder at my good fortune. Thanks, Jim. REFERENCES
FIG. 2. Professor Konrad Bloch at the Gordon Research Conference on Lipid Metabolism at Andover, N.H. June 1967, playing tennis (his opponent, not pictured, was Howard Goldfine).
chapters, and reviews primarily addressing lipid metabolism in B. megaterium. During most of this period Konrad and I kept in touch, occasionally by phone, but most often in person at scientific meetings (Fig. 2), and he never failed to ask me how our research on B. megaterium was coming along. The last time I saw him was in October of 1995 when I stopped by his office at Harvard on my way back from a P450 meeting at Woods Hole. Although by this time his memory was starting to betray him, he continued to show an interest in my research, and even indicated he had read several of our JBC papers on the mechanisms of cytochrome P450 induction by barbiturates in B. megaterium. In closing this brief history, I will not try to retrace or summarize my research 5 during the past 38 years, except to point out that almost all of it was either initiated during my two postdoctoral years with Konrad or else derived indirectly from research undertaken during that period. However, I will allow myself to reminisce once more about how it all began with a seemingly naive question I addressed in 1960 to my Ph.D. advisor, Professor James Mead. I asked him “Who is the best lipid biochemist in the world . . .? That’s the person with whom I want to carry out my 5 For those readers who may be interested, progressive summaries of my research to about 1997 can be found in a number of reviews, books, and book chapters listed in the references section (22–29).
1. Fulco, A. J., and Mead, J. F. (1959) Metabolism of essential fatty acids. III. Origin of 5,8,11-Eicosatrienoic acid in the fat deficient rat. J. Biol. Chem. 234, 1411–1416. 2. Fulco, A. J., and Mead, J. F. (1960) Metabolism of essential fatty acids. IX. The biosynthesis of the octadecadienoic acids of the rat. J. Biol. Chem. 235, 3379 –3384. 3. Fulco, A. J., and Mead, J. F. (1961) The biosynthesis of lignoceric, cerebronic and nervonic acids. J. Biol. Chem. 236, 2416 –2420. 4. Mead, J. F., and Fulco, A. J. (1961) Distribution of label from 1-C 14-acetate in brain stearic acid. Biochim. Biophys. Acta 54, 362–364. 5. Scheuerbrandt, G., Goldfine, H., Baronowsky, P. E., and Bloch, K. (1961) A novel mechanism for the biosynthesis of unsaturated fatty acids. J. Biol. Chem. 236, PC70 –71. 6. Goldfine, H., and Bloch, K. (1961) On the origin of unsaturated fatty acids in Clostridia. J. Biol. Chem. 236, 2596 –2601. 7. Khuller, G. K., and Goldfine, H. (1975) Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids. Biochemistry 14, 3642–3647. 8. Bloomfield, D. K., and Bloch, K. (1958) Biochim. Biophys. Acta 30, 220. 9. Bloomfield, D. K., and Bloch, K. (1960) J. Biol. Chem. 235, 337–345. 10. Fulco, A. J., and Bloch, K. (1962) Cofactor requirements for fatty acid desaturation in Mycobacterium phlei. Biochim. Biophys. Acta 63, 545–546. 11. Fulco, A. J., and Bloch, K. (1964) Cofactor requirements for the formation of ⌬-9-unsaturated fatty acids in Mycobacterium phlei. J. Biol. Chem. 239, 993–997. 12. Kayama, M., Tsuchiya, Y., Nevenzel, J. C., Fulco, A. J., and Mead, J. F. (1963) Incorporation of linolenic-1-C 14 acid into eicosapentaenoic and docosapentaenoic acids in fish. J. Am. Oil Chem. Soc. 239, 449 –502. 13. Fulco, A. J., Levy, R., and Bloch, K. (1964) The biosynthesis of ⌬-9- and ⌬-5-monounsaturated fatty acids by bacteria. J. Biol. Chem. 239, 998 –1003. 2 14. Fulco, A. J. (1965) Biosynthesis of ⌬-9-monounsaturated fatty acids in slices of the ice plant, Carpobrotus chilense. Biochim. Biophys. Acta 106, 211–212. 15. Appleman, D., Fulco, A. J., and Shugarman, P. M. (1966) Correlation of ␣-linolenate to photosynthetic O 2 production in Chlorella. Plant Physiol. 41, 3608 –3613. 16. Fulco, A. J. (1965) Chain elongation, 2-hydroxylation and decarboxylation of long chain fatty acids by yeast. J. Biol. Chem. 242, 3608 –3613. 17. Fulco, A. J. (1967) The effect of temperature on the formation of ⌬-5-unsaturated fatty acids by bacilli. Biochim. Biophys. Acta 144, 701–704. 18. Fulco, A. J. (1969) The biosynthesis of unsaturated fatty acids by bacilli. I. Temperature induction of the desaturation reaction. J. Biol. Chem. 244, 889 – 895. 19. Fulco, A. J. (1969) Bacterial biosynthesis of polyunsaturated fatty acids. Biochim. Biophys. Acta 187, 169 –171. 20. Fulco, A. J. (1970) The biosynthesis of unsaturated fatty acids by bacilli. II. Temperature-dependent biosynthesis of polyunsaturated fatty acids. J. Biol. Chem. 245, 2985–2990. 21. Fulco, A. J. (1970) Temperature-mediated hyperinduction of
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22. 23.
24.
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fatty acid desaturating enzyme. Biochim. Biophys. Acta 218, 558 –560. Fulco, A. J. (1974) Metabolic alterations of fatty acids. Annu. Rev. Biochem. 43, 215–241. Mead, J. F., and Fulco, A. J. (1976) The Unsaturated and Polyunsaturated Fatty Acids in Health and Disease, Thomas, Springfield, IL. Fulco, A. J. (1977) Fatty acid desaturation in microorganisms. In Polyunsaturated Fatty Acids (W. H. Kunau, and R. T. Holman, Eds.), Chap. 2, pp. 19 –36, American Oil Chemists’ Society. Fulco, A. J., and Fujii, D. K. (1980) Adaptive regulation of membrane lipid biosynthesis in bacilli by environmental temperature. In Membrane Fluidity: Biophysical Techniques and Cellu-
26. 27. 28.
29.
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lar Regulation (M. Kates, and A. Kuksis, Eds.), Part II, pp. 77–98, Humana Press, Clifton, NJ. Fulco, A. J. (1983) Fatty acid metabolism in bacteria. Prog. Lipid Res. 22, 133–160. Fulco, A. J. (1984) Regulation and pathways of membrane lipid biosynthesis in bacilli. Biomembranes 12, 303–327. Fulco, A. J. (1991) P450 BM-3 and other inducible bacterial P450 cytochromes: Biochemistry and regulation. Annu. Rev. Pharm. Toxicol. 31, 177–203. Fulco, A. J. (1998) Barbiturate-inducible gene expression. In Toxicant–Receptor Interactions and Modulation of Gene Expression (M. S. Denison, and W. G. Helferich, Eds.), pp. 103–132 (Chap. 5) Taylor and Francis, Philadelphia.