LUMINOUS PURSUIT:
JELLYFISH, GFP, AND THE UNFORESEEN PATH TO THE NOBEL PRIZE

Contents

Dedication v
Preface vii

Prologue 1

Chapter 1 My Early Life: Manchuria, Sasebo, and the Beginning of the War 5
1.1 From Fukuchiyama to Sasebo 5
1.2 Shimomura Family Traditions 8
1.3 In Manchuria 12
1.4 To Sasebo Again 15
1.5 In Osaka 20

Chapter 2 The War Strikes Home: Ohmura Naval Aircraft Factory and the Atomic Bomb 23
2.1 Student Mobilization Order at Isahaya Middle School 24

Probably nobody thought about our schooling under such unsettled conditions, in which we couldn’t predict our fate even for one day ahead. Therefore, I went to the factory every day even before it began to function. If, as frequently happened, I found nothing useful to do, I often lay down in a sweet-potato field nearby to watch the large formations of B-29s flying east, high above Mount Taradake. It was beautiful to see the B-29s shining silver against a background of blue autumn sky. About ten minutes after the bombers had passed by, we used to see black smoke in the Ohmuta industrial area located on the opposite shore of the Sea of Ariake, and we could only imagine the scenes of carnage over there.

2.2 The Atomic Bomb on Nagasaki 27

I saw a single B-29 going from north to south toward Nagasaki, about ten miles away, and wondered because its course was unusual. The B-29 dropped two or three parachutes and I heard sporadic gunfire. I watched the parachutes carefully, but could not see any people hanging from them. Within minutes, another B-29 followed the first one on the same course, then a siren sounded the “all clear.”
　We returned to our factory building, expecting to resume work. As soon as I sat down on my work stool, a powerful flash of light hit us through the small windows. We were blinded and unable to see anything for about thirty seconds. Then, maybe forty seconds after the flash, we heard a loud sound and felt a sudden change of air pressure. We were sure that a huge explosion had occurred somewhere nearby, but we didn’t yet know where.

The sky rapidly filled with dark clouds, and when I left the factory to return home, a drizzling rain had started; it was black rain, a mixture of ash, water, and nuclear fallout. When I arrived home an hour later, my white shirt had turned completely gray. My grandmother took one look at me and quickly readied a bath so that I could clean myself. That bath may have saved me from radiation poisoning caused by the fallout in the black rain.

2.3 After the War: Searching for a Future 29
2.4 After the War: Disappointments 32

Chapter 3 Struggling to Learn: Nagasaki, Nagoya, and Luciferin (1948 – 57) 35
3.1 Nagasaki Pharmacy College and Nagasaki University 35
3.2 The Hirata Lab and Cypridina Luciferin 41

On my first day at the Hirata lab, Professor Hirata produced a large vacuum desiccator from somewhere and told me, “This contains dried Cypridina (sea firefly).” They were crustaceans each about the size of a sesame seed. Then he explained how Cypridina emits light through the interaction of an organic compound called luciferin and an enzyme luciferase, that the luciferin is extremely unstable and decomposes in the presence of oxygen, and that Professor Newton Harvey of Princeton University had been trying to purify the luciferin for the past twenty years but had not successfully obtained a pure luciferin suitable for structural study. Harvey visited Japan in 1916 during his honeymoon and concluded that Cypridina was a good research material for the study of bioluminescence. Since then, he had continued his study using material obtained from Japan.

3.3 An Unexpected Success: Luciferin Crystals

I continued to extract and purify luciferin about once a month, because I could not obtain any crystals, no matter how hard I tried. However, the luciferin was finally crystallized by accident on a cold day in February 1956.

The night before, I was trying to crystallize the luciferin as usual, but I had exhausted all my ideas by about 10:00, although there was a small amount of purified luciferin left. I decided to use it in amino acid analysis. I added an equal amount of concentrated hydrochloric acid to the luciferin sample. The yellow color of the sample instantly changed to a dark red. Since an oven was not ready, I decided to heat the sample the next morning. I left the dark red solution on a shelf and went home. When I came back in the morning, the solution was colorless. I thought that the sample was hydrolyzed, but upon close inspection I found a tiny amount of black precipitate at the bottom of the test tube. Under the microscope, this precipitate could be seen as red needle-shaped crystals.

My success in crystallizing the luciferin was accidental: however, I had accomplished what the Princeton researchers couldn’t, and it gave me hope for the future, which had seemed dark since the end of the war. I was so happy that I couldn’t sleep for a couple of days. However, the most important reward I received was probably the self-confident I gained, that any complex problem could eventually be solved by effort.

Chapter 4 To America and Back: Marriage, Princeton, and Discovery of Aequorin and GFP 51

I recall that the Hirata lab was a wonderful laboratory maintaining a splendid atmosphere. Nobody taught me anything formally there, but I learned many things by observing other people and was then able to develop various techniques myself. Only once did Professor Hirata tell me, “Mr. Shimomura. Mass spectrum can tell you an exact molecular weight, not the approximate one,” but I no longer remember the circumstances under which he instructed me about that.

4.1 Marriage with Akemi: Departure from Japan 53

On August 27, 1960, I left Yokohama on the ocean liner Hikawa-maru for Seattle with over 200 Fulbright fellow scholars and students. It was my thirty-second birthday. I had just gotten married. Because it was also the las Pacific cruise of Hikawa-maru, the pier was completely filled with people. (Hikawa-maru had made her first trip to Seattle thirty years earlier, and she had weathered the war as a hospital ship, while her sister-ships had been destroyed.) My new wife, her mother, and some of my friends were among the crowd. There were thousands of festive colored tapes between people on the boat and the people on the pier, connecting them. I will never forget the vivid scene as the ship began to move and numerous tapes started to break and then fall down toward the water.

4.2 To Princeton 59

When I first visited Dr. Johnson’s room, he produced a small vial filled with white powder, explaining, “This is the freeze-dried light organs of the luminous jellyfish Aequorea, and it should emit light when mixed with water.” We went into a dark room to test it, but we could not observe any light. However, he enthusiastically described to me how abundant Aequorea were in the sea at Friday Harbor, in Washington state, and described how brilliantly luminous they were. Then he asked me if I was interested in studying its bioluminescence. I didn’t know anything about the jellyfish, but was eager to study a new luminous organism, so I answered, “I will be glad to do it.” Thus, we agreed to travel to Friday Harbor the following June.

4.3 Friday Harbor 65
4.4 The Jellyfish Aequorea 71

The Aequorea were extremely abundant. We would see a stream of floating jellyfish sweeping by alongside the lab dock in the early mornings and evenings, riding upon currents caused by the tides. We carefully scooped up the larger ones into buckets, on by one, using a shallow dip-net to avoid damaging them. A specimen of Aequorea is formed like a hemispherical umbrella, nearly transparent, with faint radiating lines along the outer half. An average specimen measures 7–8 centimeters in diameter and weighs about 50 grams.
　Aequorea, when stimulated, emits light along the edge of the umbrella, displaying a green ring in darkness. Because its luminous organs are located only along the edge, we cut off the margin of the umbrella, making a strip of 2–3 millimeters in width, and we called this a ring. We used the rings in our experiments. By squeezing about thirty rings about gauze, we obtained a turbid liquid which emitted weak light for a long time.

Often, I meditated on a drifting rowboat under the clear summer sky, so that nobody would disturb me. There was little traffic at sea around Friday Harbor at the time, and since a rowboat has the right of way over any vessel with a motor, ferries always kept a wide berth. However, if I fell asleep and the tidal currents carried the boat away, I needed to row for a long time to return to the dock. One afternoon on the boat, a thought suddenly came to me. It was quite a simple idea: Even if luciferin and luciferase are not involved in the jellyfish bioluminescence, some protein is probably involved in the bioluminescence reaction. If so, the activity of that protein would very likely to be altered or affected by a change of pH.

I threw away the luminescing solution into a sink. The inside of the sink lit up instantly with bright blue light! Because there was seawater flowing into the sink, I suspected that seawater had triggered the reaction. Since the composition of seawater is well known, I quickly determined that calcium ions had caused the sudden luminescence. It was sheer luck that some seawater was in the sink at the right time.

4.5 The Busy Year of 1962: Finding the First Traces of GFP 74

During the column chromatography of aequorin, we found a trace amount of protein that showed green fluorescence and eluted sooner than aequorin, and we also purified it. The protein is now called green fluorescent protein, or GFP, and its fame and applications have far exceeded those of aequorin.

4.6 Fireworms in Bermuda 77

On August 10, 1962, we flew to Bermuda to study the fireworm Odontosyllis, a segmented sea-dwelling worm that employs luminescence in its mating and reproduction. The female worm’s green light attracts male worms to her. This creature may have been the source of the light display seen by Christopher Columbus’s expedition in 1492, and interpreted by the sailors as an indication of land, as they voyaged through the Bahamas.

Odontosyllis is a 1–2 centimeter long worm that comes up to the surface of the sea for only a few minutes, one hour after sunset, and only for a few days after each full moon. First a female worm emitting a green light shows up on the surface and makes small circles; then a few seconds later, a number of male worms appear and rush toward the female worm. Their activities look like green fireworks, except that the movement is in the opposite direction, and it happens all over the surface of the sea at the same time.

Chapter 5 A Risky Endeavor, Raw Jellyfish, and Mysteries of Bioluminescence 81
5.1 Back in Nagoya: 1963 – 65 81
5.2 Glow Worms in New Zealand 83

In February 1965, I traveled to New Zealand to study two bioluminescent organisms, the cave worm Arachnocampa and the freshwater limpet Latia, with research funds from JSPS (Japan Society for the Promotion of Science). I went together with Dr. Yata Haneda, the director of the Yokosuka City Museum. We arrived in Wellington, New Zealand, via Hong Kong and Sydney, and then rented a car and drove toward Auckland. On the way, we briefly investigated a giant luminous earthworm at Palmerstone North, and saw the glow worm Arachnocampa at the famous cave there, although we were not permitted to collect any because the glow worm cave was an important tourist attraction.

5.3 Back to Princeton: My Family Grows 87
5.4 Luminescence Mechanism of Aequorin: Friday Harbor 95

Why did I decide to invest my time in studying aequorin? The explanation involves understanding the general principles of bioluminescence. While people have been intrigued by living creatures that give off light—bioluminescence—since ancient times, much remains to be understood about the functions, chemical processes, and evolutionary paths involved. Sometimes, the chemical processes give hints as to why and how the ability to emit light developed, and may also suggest useful applications for scientific research or medical study.
　The overall process of bioluminescence involves the release of energy, as a bright burst of light, in a chemical reaction such as the oxidation of a molecule. The light-releasing molecule is termed a “luciferin,” named after the Latin for “light-bearing” as in the name of the fallen angel Lucifer. The term “lucifer” had become a common noun for a friction match prior to the coining of the chemical term late in the nineteenth century. To early scientists, the bioluminescence may have resembled the lighting of a match or candle. These luciferins can differ among different organisms, but some unrelated species utilize the same luciferin. To understand the chemical process of bioluminescence requires knowing the structure of the molecule that is broken down or oxidized to give forth light, how it is acquired (such as by eating smaller sea creatures that produce the luciferin) and where it is stored, the substance (enzyme) that causes that light-emitting reaction to occur or continue, and the factors that trigger the process. The trigger can be an environmental change or some ion released by the bioluminescent creature to control the light given forth. Many ocean species display luminescence; the reason is often not known. In addition, the color of their light may be affected by a fluorescent protein. All these aspects of bioluminescence can yield interesting insights into nature, and possibly useful applications, if they do not remain utterly mysterious.

5.5 How Do You Prepare Jellyfish? 97

In 1974, I ultimately concluded that aequorin bioluminescence and Cypridina bioluminescence employed the same type of chemical reaction, and drew the structure of the light-emitting group of aequorin based on the structure of Cypridina luciferin. In 1975, I named the light-emitting group of aequorin “coelenterazine” and the acylated AF350 “coelenteramide.” I would like to emphasize here that the structure of coelenterazine was conceived because the chemistry of the Cypridina luminescence had been known already.

5.6 My Research on Subjects Other Than Aequorin: 1965 – 1978 103

Starting work in 1966, I elucidated the structure of the luciferin of the New Zealand fresh water limpet Latia, and investigated the properties of Latia luciferase.

I visited Kristinebergs Zoological Station in Sweden together with Dr. Johnson in August 1966 to study the bioluminescence of the krill Meganyctiphanes. Krill are tiny shrimp, 2–3 cm long, with ten small light organs that emit very intense blue light. They live deep in the sea in the daytime, but come up near the surface at night. We stayed at the laboratory at the Station for one month, but we obtained only 150 specimens, and thus could not do a detailed study. Instead, we enjoyed the beautiful seashore and sightseeing of historic Viking remains.

We extracted a bioluminescent substance from the material [Chaetopterus] we obtained in 1965 in Los Angeles. But the extract was slimy and viscous, and difficult to purify, probably because it contained a high concentration of nucleic acids. Upon finally completing purification and leaving the purified material standing for a while, however, we found that it gradually crystallized by itself. We studied its properties and learned that it was a bioluminescent protein like aequorin, that is, neither a luciferin nor a luciferase. We still lack full information concerning the nature of its light-emitting chromophore.

I went to Uozu, Japan with Dr. Inoue, of Meiji University, in 1970, to study the bioluminescence of Watasenia; I returned to Uozu in 1974. Dr. Inoue was a good friend of mine and also of my dear friend Dr. Goto, so I was happy to have this chance to collaborate with him. Watasenia is a small deep-sea squid, about 5 centimeters long. The squid comes to the shallows by the shore in April and May to lay its eggs. Its scientific genus name Watasenia was derived from the name of Shosaburo Watase who first described this squid in detail.

5.7 Photoprotein 108

Chapter 6 From Mushrooms in Woods Hole to Red Jellyfish in Bergen 111
6.1 Dr. Johnson’s Retirement: Our Move to MBL (1977 – 81) 112

In 1962, we discovered the green fluorescent protein GFP, and reported the properties of GFP in 1974. In 1979, we were finally able to study the chromophore of GFP.

In 1989, the structure of dinoflagellate luciferin was determined by Dr. Nakamura, who had also determined the structure of krill luciferin a year before.

In 1980, Dr. Marie-Therese Nicolas of France extracted and purified a bioluminescent substance from scale worms at my lab and obtained a protein that emitted light in the presence of superoxide anions. Scale worms are about 2 centimeters long, with the whole body covered by scales. They are clever worms, well adapted for survival. When attacked by other animals, a scale worm can detach two or three scales from its own body and escape to other places, leaving behind the luminous scales as a target for the predator. Scale worms can regenerate their lost scales.

6.2 At the Marine Biological Laboratory in Woods Hole 115
6.3 Tsutomu and Sachi Leave Home 118

We started to study a luminous millipede called Luminodesmus (also known as Motyxia) in 1980. The millipede is about 2 centimeters long, and inhabits the Sierra Nevada Mountains in California, at about an altitude on 1500 meters, and shows up on the ground under the giant Sequoia trees just after the snow has melted away, usually in the period between mid-April and early May. Since moonlight impedes locating the luminous millipedes, we chose a period of the new moon for our collection trip.
We studied the luminous brittle star Ophiopsila in collaboration with Dr. Paul Brehm, a professor at the State University of New York at Stony Brook at that time. Ophiopsila is a brownish brittle star, with five snaking thin arms of 5 centimeters long, and abundantly seen on the shores of Catalina Island near Los Angeles.

6.4 My Friend Goto 128

Chapter 7 The 1990s: Losses, Gains, And the Rise of GFP 131
7.1 Dr. Johnsons’s Passing 131
7.2 The Passing of Relatives in Japan 133

When I was fifty-seven years old, I privately planned that I would stop my study of bioluminescence fifteen years later in the year 2000, because I wanted to write and publish a book on bioluminescence for future generations of researchers while I had the energy to do it. Thus, I chose the study of luminous mushrooms as my last major project.

One reason I chose to study mushrooms was because we had found so many species growing near our house. I had become curious about them. We discovered that the oak logs left from clearing land for our house would readily grow both wild and cultivated kinds of fungi, so we had cultivated our won shiitake mushrooms. We saw mushrooms everywhere as we tended our property or walked in the nearby wooded areas; some, I learned, were known to be luminous.

GFP became well known to the general public by the appearance of fluorescent animals, such as a Medaka fish, mouse, frog, and rabbit. The most famous of them was probably the rabbit named Alba that was created at a Paris laboratory by order of a Chicago artist who wanted to display the animals as an object of art. However, it brought on public criticism, and the laboratory refused to hand the animal over to the artist. Fluorescent Medaka fish and zebra fish are produced in Taiwan and being sold widely. There are various problems in producing such fluorescent animals, including obvious ethical issues, and it seems doubtful that this application will contribute much to the welfare of mankind of the progress of science.

7.3 The Value of Pure Science 139
7.4 Norway and the Jellyfish Periphylla 140

The bioluminescent jellyfish Periphylla is widely distributed in the world. Its size is usually several centimeters in diameter, not so different from Aequorea. In certain Norwegian fjords, however, they grow to over eight inches in diameter, weighing nearly two pounds. Because the entrances of fjords are narrow and shallow, their environment probably differs significantly from the open ocean. I began to study this jellyfish in 1996 using specimens given to me by Dr. Par Flood of Norway, and I quickly found that its luminescence was caused by coelenterazine and a luciferase. This luciferase, however, was an interesting enzyme with an unusually high activity level.

Chapter 8 Going Full Circle: Nature’s Gift, Scientific Honors, and Revisiting Japan 145
8.1 After My Retirement 145

The Pearse Prize Lecture Session was held at the Twelfth International Congress of Histochemistry and Cytochemistry (ICHC) meeting at the University of California in San Diego. The title of my lecture was “The discovery of aequorin and green fluorescent protein.” Next, I was invited to the International Bioluminescence and Chemiluminescence Symposium at Yokohama, Japan in early August and gave a special lecture titled “Aequorin & GFP: An historical account.”

8.2 Nature’s Gift: Abundance and Disappearance of Aequorea 148

I would like to add a bittersweet mention here regarding Aequorea. We traveled to Friday Harbor nineteen times to collect the jellyfish from 1961 until 1988, obtaining a total of about 850,000 specimens of Aequorea.

Mysteriously, however, the jellyfish population decreased drastically since then, so that they were rarely seen after 1990, and we experienced difficulty in collecting even a few specimens. This depletion of the jellyfish population was not a result of scientific study. We had rarely collected jellyfish smaller than about 7–8 centimeters in diameter for our research, since these were too small to process efficiently. Also, we and other scientists who studied jellyfish regularly witnessed so many washing by on the currents that we could only collect a small fraction of the larger ones. The sudden drastic decrease may have resulted from the environmental contamination of the sea bed by crude oil spilled by the Exxon tanker Valdez near Alaskan shores in 1989, or from the warming of the earth, or it could have resulted from other causes yet unknown.

Chapter 9 The End of the Path: The Nobel Prize Announcement and Nobel Week 157
9.1 Preparing for Stockholm; the Order of Culture Award 158

“[…] You are now honorary professor of physiology and biophysics.” Thus, it meant that I was suddenly an honorary professor since several years ago! My retirement [from Boston University], which had happened without anyone seeming to take much notice at the time, was now the occasion for this honorary position. Perhaps, though, it was an appropriate continuation for my path. After all, I had acquired my first PhD in Japan without embarking deliberately on the degree program, but rather by being offered it incidentally as a result of my research and the Fulbright Fellowship. So my entire career was, in that way, illogical.

9.2 To Stockholm 164
9.3 The Nobel Week Begins 166
9.4 The Nobel Prize Ceremony and the Nobel Banquet 170
9.5 The End of Nobel Week 173

Chapter 10 After Stockholm: Visiting Los Alamos, Remembering Nagasaki 177
10.1 My Welcome in Japan 178

While at Nagoya I was able to visit the cemetery of Professor Hirata with his widow and two daughters. When I put my head low in front of his tombstone, I felt as if I heard him speaking in his usual gentle voice with a smile, saying to me, “You did well.”

10.2 Lindau, Germany 180
10.3 Research in Russia 182
10.4 An Invitation to Los Alamos, Where the Atomic Bomb was Built 185
10.5 Fragments of History? 188

On the unforgettable day—August 9, 1945—I saw the first B-29 that dropped a parachute with an instrument, probably to collect the explosion data. When the second B-29 had reached a position above Nagasaki, I had gone back into the building, and sat down on my stool. At that moment I was assailed by extremely strong flash of light. If I had continued to look at the sky, it probably would have damaged my eyes.
　Here, at Los Alamos, I saw the same parachute with the instrument on display that I had seen seventy years ago against a clear blue sky. There was an explanation of the explosion mechanism of the atomic bomb given, as if there was no secret involved. It seemed as if it could be made almost anywhere if the materials were available; I was a little frightened at the idea.
　In Nagasaki in 1945, it had been days before people learned what the bomb had been. Details were not publically known, and even the extent of the damage was hard for people to assess because of disruptions to transportation and communications. Afterward, the injuries to people and the dangers of radiation were poorly understood because there had been no atomic bombs before. No one knew the full consequences. More than half a century later, Japan was still collecting data on the long-term effects: Periodically, when we visited Japan, Akemi was supported to see doctors who were continuing to trace the health conditions and history of people within a certain radius of the impact site.
　How much do we know, and how much do we really understand? I wondered, as we visited this place where science had accomplished a terrible wonder. John Markoff wrote a story for the New York Times (May 12, 2013) about our visit. In his story, he noted Dr. Pearson’s comment that the person who started explaining the instrumentation attached to the parachute was dumbfounded to hear that I had actually seen the drop of the instruments. Perhaps they are just old history preserved in a museum, for many people, even scientists, who live in a post-atomic world.

10.6 Akemi’s Memories of the Atomic Bomb 193
10.7 Science and War: Back to Nagasaki 198

The next year, I returned again to Japan. One of the events I was invited to was the Pugwash Conference. In January of 2015 I had received a letter from the organizer of the 61st Pugwash Conference on Science and World Affairs inviting me to participate in it. It was interesting to me because it would be held at Nagasaki in November of that year, upon the 70th anniversary of the atomic bomb. It was titled “Nagasaki’s Voice: Remember Your Humanity,” and called for the abandonment of nuclear weapons. The Pugwash Conferences’ efforts had been recognized by the 1995 Nobel Peace Prize, and many Nobel laureates have supported their call for a world without the horrors of nuclear destruction.

Science, solitary or otherwise, continually connects back to the world in unforeseen paths. After this event I felt my life as a chemist had converged to an appropriate end: I started my life of being a chemist because of the atomic bomb at Nagasaki 70 years ago and now in this Pugwash Conference I realized I had had long enough of a life as a chemist.

Chapter 11 Epilogue 203
11.1 Was the Discovery of GFP Good Luck? 204
11.2 My Research Life and Communications with Other Scientists 204
11.3 The People I Respect 206
11.4 A World of Thanks 207

Selected Bibliography 209
Index 211

 

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Excerpt from Osamu Shimomura, Sachi Shimomura, John H Brinegar, Luminous Pursuit: Jellyfish, GFP, and the Unforeseen Path to the Nobel Prize (Singapore: World Scientific Publishing, 2017).

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