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h u m a n s since 1968, we have sequenced the hemagglutinin from A/duck/Ukraine/63 (H3N8). The very close homology (96% at the amino acid level) of its primary structure with that of the fn'st strain carrying the H3 hemagglutinin in m a n strongly suggests a recent c o m m o n ancestry for both hemagglutinins '23. However, other mechanisms must exist. The reappearance of the H1N1 virus in 1977 after a 20-year absence shows that subtypes previously circulating in m a n can suddenly reappear from an apparently frozen or cryptic state 24. Finally, a general question that has remained unanswered so far is: why do shifts always occur in China? Future d e v e l o p m e n t s

Influenza vaccines now in use are, in fact, not designed to overcome the variability of the virus; the vaccine composition is simply changed following the drift observed during the previous season. Moreover, no attempts are made to fight shift. However, the recent explosion of structural information on influenza surface antigens, and especially on the hemagglutinin, can form a new starting point for future developments. Recently, it has become technically feasible to synthesize peptides of reasonable length (up to about 40 residues) efficiently. Somewhat unexpectedly, it turns out that peptides, even those that do not correspond to immunodominant regions of the surface of a protein molecule, can elicit antibodies reactive with the native molecule. In the case of influenza HA, this offers the possibility o f trying out peptides from conserved regions and assessing their immunogenic, virus-neutralizing and animal protective potency. It turns out that in certain cases influenza A strains (belonging to different subtypes) and even influenza B virus, can be neutralized. There is some preliminary evidence that in some cases, protection of tested animals ensued 25. Although potentially promising, this approach still requires considerable refinement with respect to dose, adjuvants and carders before it can be applied on a large scale to humans. Another approach is to manipulate the antibody repertoire by modifying the viral antigens using genetic engineering techniques. Influenza hemagglutinin antigenic determinants have already been expressed in bacteria and in animal cells (see for example Refs 26 and 27). Using these systems, it might be easier to preserve a sufficient level of immunogenicity in the protein preparation to be o f practical use. Other strategies aimed at overcoming the disease might be based on inhibition of a specific and conserved stage of the life cycle of the influenza virus. New (or more effective) c o m p o u n d s might be found

Gerhard, W. Cell 31,417-427 12 Blok, J. and Air, G. M. (1982) Virology 121, 211-229 13 Soeda, E., Mamyama, T., Arrand, J. R. and Griffin, B. E. (1980)Nature 285, 165-167 14 Both,G. W. and Sleigh, M. J. ( 1981) J. Virol. 39, 663---672 15 Huddleston, J. A. and Brownlee, G. G. (1982) Nucl. Acids Res. 10, 1029-1038 16 Laver W. G., Air, G. M., Webster, R. G. and Markoff, L. J. (1982) Virology 122,450-460 17 Staudt, L. M. and Gerhard, W. (1983) J. Exp. Med. 157,687-704 18 Wiley, D. C.. Wilson, 1. A. and Skehel, J. J. References 1 winter, G., Fields, S. and Brownlee, G. G. ( 1981) (1981 ) Nature 289, 373-378 19 Underwood, P. A. (1982)J. Gen. Virol. 62, Nature 292, 72-75 2 Van Rompuy, L., Min Jou, W., Huylebroeck, D. 153-169 and Fiefs, W. (1982)J. Mol. Biol. 161, 1-11 20 Colman, P. M., Varghese, J. N. and Laver, W. G. 3 Webster, R. G., Laver, W. G., Air, G. M. and (1983)Nature 303, 41--44 Schild, G. C. (1982)Nature 296, 115-121 21 Webster, R. G. and Laver, W. G. (1975) in The 4 Markoff, L. and Lai, C. J. (1982) Virology 119, Influenza Viruses and Influenza (Kilbourne, 288-297 E. D., ed.), pp. 269-314, Academic Press 5 Bentley, D. K. and Brownlee, G. G. (1982)Nuel. 22 Scholtissek, C., Rohde, W., Von Hoyningen, V. Acids Res. 10, 5033-5042 and Rott, R. ( 1978) Virology 87, 13-20 6 Ward, C. W., Elleman, C. and Azad, A. A. ( 1982) 23 Fang, R-X., Min Jou, W., Huylebroeck, D., Biochem. J. 207, 91-95 Devos, R. and Fiefs, W. (1981) Cell 25,315-323 7 Elleman, T. C., Azad, A. A. and Ward, C. W. 24 Palese, P. (1980) Trends Biochem. Sci. 5(3), (1982) Nuel. Acids Res. 10, 7005-7015 III-V 8 Krystal, M., Elliot, R. M., Benz, E. W. Jr, 25 Sutcliffe, J. G., Shinnick, T. M., Green, N. and Young, J. F. and Palese, P. (1982) Proc. Natl Lerner, R. A. (1983)Science 219, 660--666 Acad. Sci. USA 79, 4800-4804 26 Davis, A. R., Bos, T., Ueda, M., Nayak, D. P., 9 Shaw, M., Lamb, R. A., Erickson, B. W., Dowbenka, D. and Compans, R. W. (1983) Gene Briedis, D. J. and Choppin, P. W. 0982) Proc. 21,273-284 Natl Acad. Sci. USA 79, 6817-6821 27 Gething, M.-J. and Sambrook, J. (1981) Nature 10 Vehocyen,M., Fang, R.-X., Min Jou, W., Devos, 293,620-625 R., Huylebrocck, D., Saman, E. and Fiefs, W. 28 Vcrhoyen, M.. Van Rompuy. L.. Min Jou. W.. (1980) Nature 286, 771-776 Huylebroeck, D. and Fiers, W. (1983) Nucl. 11 Caton, A., Brownlee, G. G., Yewdell, J. W. and Acids Res. 11 4703-4712

which specifically interfere with adsorption of the virus, with uncoating the viral R N A (e.g. weak bases raising the intralysosomal pH), with the influenza virus-specific R N A replicase system, etc. Whatever approach m a y ultimately lead to an effective control for influenza in h u m a n s we m a y hope that the developments in recent years have brought us considerably closer to a successful solution.

50 Years Ago Progesterone Karl H. Slotta It was really presumptuous of me to start studying chemistry in 1919. In 1914 a French bullet had gone straight through m y chest a n d in 1915 a piece of shell b e c a m e lodged in m y brain, often causing seizures. M y education in t h e classics - Latin and G r e e k - h a d gone down the drain, m y k n o w l e d g e of m a t h e m a t i c s was below zero, a n d I h a d n e v e r had a single class in chemistry. W h e n I was interviewed by Heinrich Biltz, the Director of the Chemical Institute at the old and v e n e r a t e d University of Breslau, I wore m y l i e u t e n a n t ' s u n i f o r m with a row of medals - a n d I probably clicked m y heels on entering his office. H e inquired a b o u t m y b a c k g r o u n d in c h e m Karl H. Slotta is Emeritus Professor of Biochemistry and Medicine, University of Miami School of Medicine, Miami, FL. USA.

istry and on hearing that t h e r e was n o n e , chuckled happily: ' T h a t ' s great! T h e n you are not b u r d e n e d by any w r o n g concepts.' I was accepted and started m y first university t e r m , aged 24. Studying was like an obstacle course in the years after W o r l d W a r I, r u s h i n g from one e x a m to the o t h e r to m a k e up for lost years. T h e r e was a lack of reagents a n d glassware, n o b o d y h a d any m o n e y , and inflation was soaring. L o o k ing back, it s e e m s a miracle that I m a n a g e d to get b o t h m y P h D a n d m y first job as Biltz's private assistant by 1923. In due course I b e c a m e 'Oberassistent' of t h e C h e m i c a l Institute, was given the right to s p o n s o r P h D dissertations (1927), to lecture ('Privatdozent', 1929) and finally received the title 'Professor' in 1935. In 1925, m y future father-in-law,

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418 Ludwig Fraenkel - Chairman of the Obstetrics and Gynecology Department drew my attention to the publication by two young gynecologists in Dresden. They claimed to have found a reliable test to predict the sex of a fetus. Together with two gynecologists in Fraenkel's department I followed up this study, and we came to the conclusion that it had no scientific foundation. With youthful lack of diplomacy I said as much at a meeting of the Breslau Gynecological Society, and my medical audience reacted angrily to this impudent chemical upstart. Some time later, however, the authors themselves dropped their original claim of reliability and I was pardoned. In the following years, with the help of twenty PhD candidates, I tried to clarify half the problems in organic chemistry: isocyanates, biguanides, tyramine, mescaline, azo-indicators, hydrocupreone and papaverine were on the extensive menu. In 1931, I wrote a book, ArzneistoffSynthese (Synthesis o f Drugs), hoping to relieve a suffering humanity and to become an overnight millionaire. Unfortunately, nothing of the kind happened, especially as a translation was sold in the US without regard to my rights as author, being considered the property of the Nazis after World War II. During our frequent evening walks my father-in-law introduced me to his ideas about the female sex hormone present in the corpus luteum. Having found in 1903 that this organ is an endocrine gland, he was convinced that it contained a hormone of great importance for the maintenance of pregnancy. He finally talked me into attacking this difficult problem, and when Heinrich Ruschig asked me to direct his PhD research in 1930, the right co-worker had been found: gifted, dedicated, interested, and possessing abundant energy. Our starting material was sows' ovaries from slaughter houses. At that time the University Women's Hospital offered shelter and light employment to indigent pregnant women (the 'Hausschwangeren'), and soon these sat in groups and cut the corpora lutea out of the sow's ovaries. Unfortunately, this material had a terrible smell which caused innumerable complaints throughout the hospital, while my fatherin-law complained more and more about the costs. The smell from the processing of the corpora lutea mass also caused an uproar at the Chemical Institute, and its director bemoaned the high cost of ~eagents. Luckily, we soon received financial help from the 'Notgemeinschaft Deutscher Wissenschaft' and later also

T1BS - November 1983

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later known as progesterone, active in doses of 1.5 mg. One of our major concerns at that time was the magnitude of the required dose. Nobody knew that the ratio of estrone to progesterone within the menstrual cycle of a woman is 1 : 100! Soon we had even more serious problems: Hitler had come to power on 30 January 1933 and we felt storm clouds gathering which threatened our troika. Afraid that time would run out, we worked feverishly. Direct purification of the crystalline fractions could not be achieved with the methods available to us in the thirties. The final isolation of the hormone had to be made with a functional derivative. Attempts at acetylation, reduction and addition to the double bond were of no avail. Finally, on 3 May 1933, Ruschig tried to treat our oil with dinitrophenylhydrazine in methanol. Half an hour later lovely red needles precipitated. It was possible to reform them to the active substance by reacting them with pyruvic acid. The same results were obtained by letting the alcoholic solution stand with semicarbazide. The semicarbazones fell out with water and were hydrolysed with 80% oxalic acid on a waterbath. After diluting the oxalic acid solution with water, a precipitate separated which proved to be mainly luteosterone A. Luteosterone C (progesterone) was obtained from the colloidal filtrate in two modifications: m.p. 121-122°C and 128°C (mixed m.p. 126°C). The isolation and purification of the corpus luteum hormone by means of keto reagents was reported to I. G. Farbenindustrie Hoechst in two memoranda, dated 7 June 1933 and 1 February 1934. Both documents have been preserved. They include melting points, analytical data and molecular formula. Our paper on the isolation of the corpus luteum hormone, with a reproduction of the coffin-shaped crystals, was published in the July issue of Berichte der Deutschen Chemischen Gesellschaft (Slotta, K. H., Ruschig, H. and Fels, E., Ber. 67, 1270). During the same year, the purification of the hormone was also reported by three other laboratories*. The quantities of luteosterone C (progesterone) obtained by the method developed in 1933 were sufficient to determine its constitution: the molecule contained two carbonyl groups and a double

from I. G. Farbenindustrie Hoechst. Whatever Ruschig and I, the chemists, achieved was in large measure due to the inspiring and tireless research of our medical colleague, Erich Fels. L. Fraenkel suggested that he join our team, as he considered him his most dedicated and scientifically gifted collaborator. Finding the Clauberg test unsatisfactory, Fels turned to the Corner test. This is based on the observation that mating of a rabbit in season results in its uterus becoming sensitized by follicular hormone; subsequently corpora lutea are formed in the ovaries and pregnancy occurs. This 'progestational' change of the uterus is suppressed, however, if the ovaries are removed 16-20 hours after mating. By administering sufficient quantities of corpus luteum hormone over the following 5 days, this effect can be reversed. During the next 4 years Fels performed operations on over 800 rabbits. Needless to say, this required considerable surgical skills; moreover, the exact, quantitative evaluation of the test results called for extensive histological experience. The isolation of the corpus luteum hormone proved particularly difficult because, in contrast to the follicular hormone, it is insoluble in water. The follicular hormone contains a phenolic hydroxy group and can be extracted with alkali. The corpus luteum hormone, however, is destroyed in an alkaline solution. First it has to be extracted with alcohol in order to free it from a large amount of inert materials, such as triglycerides, fatty acids, cholesterol and proteins. Then the phospholipids have to be precipitated with acetone. Two separation procedures, the addition of petroleum-ether to the 70% alcoholic solution, and the extraction of the petroleum-ether solution with 40% alcohol, resulted in a ten-fold purification. The next step was treatment of the petroleum-ether ,solution with A1203 in order to remove the remaining inert materials; this yielded a constant and reliable preparation of 1 Corner unit per 4-5 mg. "1 Butenandt, A., Westphal, U. and Hohlweg, Two crystalline substances separated from W. (1934) Z. Physiol. 227, 84 an ice-cooled petroleum-ether solution 2 Winterstciner, O. and Allcn. W. M. (1934) J. Biol. Chem. 107.321 of this material. Substance A, later called luteosterone A, C~H~O2 m.p. 161°C, 3 Hartmann, M. and Wenstein, A. (1934) Helv. Chim. Acta 17, 1365 physiologically inactive; and substance C.

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T I B S - November 1983 bond in a-position to one of them. Its U V absorption maximum was at 243 nm. One year earlier Professor A. Neuhaus of the Breslau Mineralogical Institute had already determined the size of the molecule by means of X-ray diffraction studies and indicated its steroid nature. Thus we were able to propose a structural formula of the hormone in August of 1934. In their thoroughly referenced book of 1959, Steroids, Fieser and Fieser therefore stated: Slotta proposed a formula which very shortly was proved to be correct by partial synthesis', and Greep confirmed this in 1977 in Ann. N.Y. Acad. Sci. 286, 1: 'The structure of progesterone as proposed in August 1934 by Slotta et al. was proved correct'. An interesting epilogue to this success story took place one year later. On 2 July 1935 I was startled by a telegram: 'Did you receive my letter of June 5? Parkes, Medical Research Council, London.' My answer in the negative elicited another wire: 'Sir Henry Dale invites you to participate in hormone discussion London July 15-17. Please acknowledge receipt of this. Parkes.' I later learned that the original invitation had mentioned the League of Nations as the sponsor of this conference, but as Hitler had already taken Germany out of the League, Nazi mail censorship prevented the letter from reaching me. The next step for me as a civil servant was to obtain permission from the Ministry of Education to travel abroad. After many difficulties but with help from the Breslau University I was finally allowed to go to London 'for scientific discussions'. The conference had originally been scheduled for 1934 for the standardization of 'male and oestrus-producing hormones' with Doisy, Butenandt and Laqueur as main participants. Due to the success with the corpus hiteum hormone in 1934, Allen and I were now invited also. The first item on the agenda regarding 'our' hormone was its name. Allen, Wintersteiner and Corner had called it 'progestin'; we called it 'luteosterone'; Butenandt wavered between the two versions. On the night Sir Henry Dale had invited the members of the conference to dine at the Athenaeum Club, Butenandt and I had a double whisky before dinner and that was it: 1 agreed to attach my ' . . . sterone' to Allen's 'proge . . .', and 'progesterone', originally suggested by Martian, received Butenandt's and also Allen's blessing. The result of this christening was published jointly (Ber. 68, 1746 and Science 82, 135, 1935), and we also agreed to define 1 mg progesterone as one inter-

national corpus luteum unit. In contrast to progesterone, no agreement could be reached on a name for the follicular hormone. With his pipe clenched firmly between his teeth, Doisy refused to give up his name 'theelin'. The days in London were probably the most interesting of my scientific career, spent with such outstanding colleagues as Doisy (estrone), Miescher (steroid hormone), Laqueur (testosterone), Marrian (pregnandiol, estrioi), A . S . Parkes and Sir Henry Dale. At our last joint dinner, Laqueur suggested that on my way home I visit the scientific laboratories of Organon in Oss, Holland. Since I had only been allowed to take very little money out of Germany, I had to decline the interesting invitation. Laqueur then threw a $20 bill across the table and told me to pay it back whenever I could. Thus he made it possible for me to make this detour. In Oss I was shown the recently crystallized testosterone and a discussion of its structure ensued. I drew the structural formula as I assumed it to be on a paper napkin. Subsequently,

it turned out to be correct. Many years later Laqueur came to see me in S~o Paulo, Brazil and I repaid the $20! But back to 1935. In October I left Germany for ever and that meant the end of any progesterone research for me. The second female sex hormone had opened the way to the contraceptive pill. However, when administered orally, it disintegrated too fast. It had to be chemically altered in the -20 and -19 positions This took another 20 years and involved many outstanding scientists. In 1934 we wrote 'the corpus luteum hormone will probably some day play a significant role in the important problem of hormonal sterilization', but at that time neither we nor anybody else visualized the far-reaching social changes brought about by 'the pill' all over the world. Karl Slotta left his native Germany because of Nazi oppression in 1935. After 20 years in Brazil he joined the University of Miami School of Medicine, where he engaged in active research until the end of the academic year in which he reached his 80th birthday (1975).

Book Reviews A favourable balance on membrane bioenergetics Transport and Bioenergetics in Biomembranes edited by Ryo S a w and Yasuo Kagawa, Japan Scientific Societies Press and Plenum Press, 1982. $45.00 (xi + 250 pages) I S B N 4 762 25332 4/0 306 41282 9 Since Albert Szent-Gy6rgyi's famous little book, the word 'bioenergetics' became fashionable in the titles of both meetings and publications. If one paraphrases on the well-known statement of Crick, and defines bioenergetics as 'a branch of science on which lectures and posters are presented in international bioenergetic Meetings' then the title of the present volume corresponds exactly to its content. The topics in it cover a broad field, ranging from mitochondrial and microsomal electron transport through ATPases to bacterial and eukaryotic transport processes. In the Preface there is an explanation of how all these (not always closely related) topics were collected together to give this volume. Between 1978 and 1981 the Japanese Government sponsored a special research project on biomembranes and part of the progress made during this period has been compiled in this publication. The result is a somewhat arbitrary selection of

topics but at the same time there are some outstanding essays and reviews, which present 'the state of the art 1982'. All the chapters are based on the original research of their authors but, as judged from the final product, the editors were liberal about their mode of presentation. Some of the papers- and these are the best ones are essay-like and give a brilliant overview. Others contain detailed data of recent research and I would prefer to see them in journals. Fortunately, the essay-like parts are the majority and are decisive in the final impact the book makes on the reader. Two closely related and, fortunately, complementary chapters deal with the proton-translocating ATPases ('Structure and Function of H+-ATPase ' by Kagawa and 'Biochemistry and Molecular Biology of Proton Translocating ATPase of Escherichia coli' by Futai and Kanazawa). Kagawa surveys the structure of the F~F, ATPases and on this comparative basis emphasizes their universal nature. A particularly interesting part in his paper is the description of the lines along which further progress is expected to be made. In Futai and Kanazawa's paper, subunit structure and reconstitution in the E. coli Fo-F~ ATPase is reviewed clearly. It is the second part within this essay that I found to be the