Research Completed at LPISM in 1988 – Reproduced and Extended in 2014

The author in his laboratory at the Linus Pauling Institute of Science and Medicine. Originally published in Science Digest, June 1986.

The author in his laboratory at the Linus Pauling Institute of Science and Medicine. Originally published in Science Digest, June 1986.

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In 1987, my colleagues at the Pauling Institute in Palo Alto, colleagues at Stanford and I published a paper that clearly demonstrated that expression of a charge-altered mutant human beta-actin (glycine to aspartic acid substitution at amino acid 245; G245D) caused non-tumorigenic, immortalized human fibroblasts to form aggressive tumors in nude mice (Leavitt et al, 1987a). When these tumor-derived cells were examined, we discovered that they exhibited further elevated expression of the mutant beta-actin and these tumor-derived cells formed tumors even more rapidly – observations that were consistent with the role of this mutation in the tumorigenic phenotype. Furthermore, over-expression of mutant beta-actin was associated with down-regulation of three abundant tropomyosin isoforms in a well-documented transformation-sensitive manner (Leavitt et al, 1986; Leavitt et al, 1987a and Ng et al, 1988). These final papers were the culmination of research conducted at the Linus Pauling Institute of Science and Medicine (LPISM) from December 1981 to March 1988.

Normally when a scientific observation is never repeated it is usually not worth remembering. In this case, twenty-six years after our 1987 publication, a study was published by Schoenenberger et al. at the Biozentrum in Basel, Switzerland, that reproduced our findings in a different cell system, a rat fibroblast model (provided to them by LPISM in 1986). Furthermore, these investigators extended our findings by characterizing new aspects of abnormal behavior of the mutant beta-actin and cells that express this aberrant protein, which help to explain its potential role in cancer such as enhancement of tumor cell motility and invasiveness.

In addition to enhancement of tumor growth and alteration of cell shape, the Swiss investigators presented the following findings to clarify and support the oncogenicity of this mutation:

  1. The mutant actin stimulated formation of ruffles at the cell periphery as shown by staining of cells with an antibody that bound specifically to the mutant epitope of the mutant beta-actin (left image below)
  2. The mutant actin concentrated primarily in these ruffles (palloidin staining reveals the location of filamentous actin in stress fibers; right image below)
  3. The expression of mutant actin inhibited the tropomysin binding to filamentous actin and tropomysin did not accumulate in the ruffles
  4. Mutant actin colocalized with Rac1 (a GTPase mediator of membrane ruffling) and beta1-integrin (adhesion protein) in the ruffles

ruffles

Back-tracking several years, the discovery of this actin mutation was made in a mutagenized cell line isolated by Takeo Kakunaga at the National Cancer Institute (NCI) in 1978. During the month that his paper was published, I walked over to NCI from my lab across the street at the Bureau of Biologics (FDA) to have a chat with Takeo about using his in vitro transformed Syrian Hamster cells as a model system to identify changes in protein expression that correlated with neoplastic transformation. After describing what I wanted to do, he seemed agreeable but then casually mentioned that he had succeed in transforming human fibroblasts into tumor forming cells. I nearly fell off my chair because human cells had never been transformed in vitro before, a major problem for cancer researchers at that time.

I blurted out that we should do the work that I had proposed in his human cell model system, comparing protein expression by the transformed neoplastic cells with their normal precursor cells. My hypothesis was that this comparison would allow identification of proteins that were turned on or turned off in expression by comparing protein profiles of the most abundant 1,000 proteins expressed in these cells and resolved by high-resolution 2-D gel separation (protein profiling). My plan was to look for charge-altering mutations in proteins that might govern neoplastic transformation and tumorigenesis. A fall-back goal was to define the pattern of qualitative and quantitative changes in protein synthesis to try and get a handle on the mysterious mechanism of human cancer development. A summary of the global changes in gene expression of neoplastic human fibroblasts was published from LPISM in 1982 (Leavitt et al, 1982).

Within two weeks, in May of 1978, I was metabolically labeling the total cellular proteins (with the amino acid S-35 methionine) of the normal fibroblasts and three strains of cell lines derived from the normal culture which were immortalized, only one of which formed subcutaneous tumors in nude mice. After four hours of labeling, I prepared extracts of S-35 methionine labeled proteins from each of the four cultures and loaded 25-microliter aliquots of each sample onto the top of clear noodle-like isoelectric focusing gels (7-inch long urea-polyacrylamide gels with the thickness of thin spaghetti) which separated the complex mixture of total cellular proteins by their net charge (isoelectric point). These gels were subjected to isoelectric electrofocusing of the proteins overnight. The next morning I harvested the spaghetti-like gels, and incubated them in a detergent that would bind to the proteins to help separate them by their molecular weights in a second dimension. So, these proteins were first denatured and separated by their net charge and then, in a second dimension, separated by their size on a thin rectangular slab gel.

After about five hours of separation in the second dimension, I was soon to learn that I had separated more than 1,000 denatured protein subunits (polypeptides) by their differing charges and molecular weights. The final step before autoradiography, which revealed the full protein profile, was to fix and stain the gels to get a glimpse of the resolution of these peptide patterns. The staining of these rectangular gels revealed only the most abundant architectural cellular proteins, the largest number of which were cytoplasmic beta- and gamma-actin, at a ratio of about 2:1 in abundance, respectively. The figure below shows what quickly appeared as the gels were de-stained. In the one tumorigenic cell line, instead of seeing a 2:1 ratio of beta- to gamma-actin, a new abundant protein at about one unit charge more negatively charged (more acidic), and half of the normal beta-actin was lost. The pixilation of these three radioactive “spots” immediately suggested to me that one of the two functional genes (alleles) encoding beta-actin had mutated, possibly due to the replacement of a neutral amino acid with a negatively charged amino acid. This prediction was no mystery to me as I had demonstrated this type of electrophoretic shift in another protein a year earlier at Johns Hopkins.

mutant actin further annotated

A number of experiments were done to build the case for the beta-actin mutation, and then I wrote a letter to Klaus Weber at the Max-Planck Institute in Goettingen, Germany, asking for his help in sequencing these actins. His lab was the only one in the world sequencing actins, e.g. the four muscle forms of actins. It only took Klaus two weeks to respond affirmatively, an indication to me that he was eager. I provided him with the actin proteins from this cell line and it took a postdoctoral fellow, Joel Vandekerhkove, and Klaus a little over a year to determine the complete amino acid sequences of the mutant beta-actin and both the wildtype beta- and gamma-actins, to define the mutation that had occurred. We published the result shown above in the top journal Cell in December 1980. Four years later, my colleagues at Stanford and I published my cloning of the mutant and wildtype human beta-actin gene, and the experiments that formally proved the mutation at the level of the gene (Leavitt et al, 1984). Three years after that, we published the experiments that demonstrated the tumorigenic effect of this mutation in immortalized human fibroblasts.

The dramatic nature of this discovery was never fully appreciated, perhaps, because no other actin mutations had been reported and it took Scheonenberger, et al. twenty-six years to complete the work published in September 2013. In another recent related development, Lohr et al. reported reoccurring beta-actin mutations in a panel of tumor cell samples from patients with diffuse large B-cell lymphoma.

One interesting piece of information that came out of our initial sequencing of these actins was the degree of evolutionary conservation of human beta- and gamma-actin. These two actins differ by only four amino acids at the N-terminus, whereas the four muscle-specific human isoforms are more divergent. Comparing the sequence of actin cloned from Saccharomyces cerevisiae (yeast) with these human sequences (sequences stored at the National Center for Biotechnology Information; NCBI) reveals that yeast and human cytoplasmic actins are 92% identical in their sequences (differing by only 31 amino acids out of 375) and most of these amino acid exchanges are conservative replacements both structurally and thermodynamically. This makes these actins the most highly conserved proteins (on a par with histones H3 and H4) among the 20,000 or so known human protein sequences. This fact presents an argument for the fundamental importance of non-muscle cytoplasmic actins in eukaryotic life. It turns out that among actin sequences of all species, no replacement of the Glycine 245 has ever been documented as a result of species divergence or mammalian isoform divergence.

When we introduced the mutant beta-actin gene into a non-tumorigenic immortalized fibroblast strain by gene transfer (Leavitt et al, 1987a), we isolated a transfected clone in which the ratio of exogenous mutant beta-actin to wildtype beta- + gamma-actin was 0.88 – a 76% higher level of expression than the mutant actin in the original mutated cell line in which the mutation arose (0.5 ratio). When we isolated and cultured the cells from a tumor formed by this cell line, the ratio of exogenous mutant beta-actin to wildtype beta- + gamma-actin had increased to 1.95, indicating that about 64% of the total cytoplasmic actin was the mutated beta-actin. Whereas the initial transfectant cell line produced visible tumors at about six weeks, the tumor-derived transfectant cells expressing 64% mutant actin formed visible tumors at about 1.5 weeks. Thus, expression of this mutation was not inhibitory to cell growth.

The other surprising finding was that cell lines expressing the transfected mutant actin gene did not have higher levels of cytoplasmic actins in them because the two endogenous wildtype beta- and gamma-actin genes were coordinately down-regulated (auto-regulated) so that the relative rates of total actin synthesis remained around 30% compared to S-35 methionine incorporation into 600 surrounding non-actin polypeptides in the protein profile (Leavitt et al, 1987b). This auto-regulation phenomenon was reproduced by Minamide et al. (1997) ten years later.

Cytoskeletal rearrangement of actin microfilaments, as well as changes in composition of tropomyosin isoforms and other actin-binding proteins, have long been associated with neoplastic transformation. However, before our study, causal mutations in a cytoplasmic actin had apparently not been considered. It is perhaps consistent then that Ning et al. (2014) have recently described genetically inherited polymorphisms in the actin-bundling protein, plastin (also discovered and cloned at LPISM), that significantly affect the time of tumor recurrence in colorectal cancer after resection and chemotherapy.

During my tenure at the Pauling Institute, I felt that Dr. Pauling understood and appreciated this work and its relevance to the fundamental nature of cancer development. Progress can be slow, but ultimately true understanding of cancer will emerge from this type of research…and I predict that cytoplasmic actins and actin-binding proteins that regulate actin organization and function in the cytoskeleton will be understood to play a central role in the manifestation of the tumorigenic phenotype.

Pauling the Swimming Cheat?

The Men's Gymnasium pool at Oregon Agricultural College, ca. 1920s.  This is where Linus Pauling would have taken his required swim test. Or did he...?

The Men’s Gymnasium pool at Oregon Agricultural College, ca. 1920s. This is where Linus Pauling would have taken his required swim test. Or did he…?

An odd nugget came across our desk recently.  From a column titled “Myth of Harvard swimming rule sinks 50 yards short of truth,” published in the Pasadena Star-News and authored by Robert Rector, we read

As recently as two years ago, many schools still had the compulsory swimming requirement, among them MIT, Columbia, Bryn Mawr, Washington and Lee, Dartmouth and Notre Dame.

And like Harvard, the test has become the stuff of legends.

One such tale holds that during the 1920s, Oregon State University had such a requirement, and Linus Pauling, who would go on to win two Nobel Prizes, could not swim a stroke. It was rumored that someone donned his number and swam for him.

It is most certainly true that, during Pauling’s time and for many decades following, Oregon Agricultural College did require that it’s students learn to swim before they could graduate.  (Presumably this was in keeping with the land grant mission of attaining practical knowledge, though it is less clear why the male students took their swim lessons and tests au naturale.  Allegedly it had something to do with the college pool’s filters.)  The unsourced bit about Pauling pulling a fast one is new to us and seems unlikely.

Pauling at the Oregon coast with his cousin Rowena, 1918.

Pauling at the Oregon coast with his cousin Rowena, 1918.

Young Pauling didn’t have any qualms about bending rules that he thought silly or inconvenient.  He infamously tried out for the college track team in order to avoid OAC’s physical education class requirements, a plan that fizzled rather quickly.

The idea that Pauling did not know how to swim, however, strikes us as improbable.  For one, OAC was a pretty small community back then – just over 3,400 boys were enrolled by the time that Pauling graduated – and it would seem to have been more difficult to slip a body double past the watchful eyes of the examiners. As a four-year ROTC cadet, one also supposes that swimming may have entered his military curriculum along the way.  Pauling and his cousin Mervyn likewise spent six weeks during the summer of 1918 helping to build wooden-hulled freighters on an Oregon coast shipyard.

More telling for us, however, is this early scene from Tom Hager‘s biography, Force of Nature. It describes the exploits of a fifteen year old boy, living in Portland, Oregon, who was hellbent on outfitting his basement chemistry laboratory.

His greatest feat was the transport of a small, brick-lined electric furnace [scavenged from an abandoned smelter in Oswego, Oregon].  Since it could not be disassembled, he talked a friend into helping him haul it down to the river, where they loaded it into a borrowed canoe, paddled miles downriver, then pushed it two miles home up Hawthorne Boulevard in a wheelbarrow.

The teenaged Pauling, it would seem, either knew how to swim or was very brave. (or foolish)

Ava Helen and Linus Pauling, 1948.

Linus and Ava Helen in their Pasadena pool.

Regardless, by 1950 Pauling had definitely sorted out his water skills to the point of building a swimming pool at his Pasadena home.  Dubbed “the pool that General Chemistry built,” the feature was funded by Pauling’s book royalties and very quickly became a bit famous.  As Ken Hedberg recalled in 1995

The Paulings had built a very nice swimming pool just below the house, a feature that made the baby-sitting task a real pleasure in the warm Pasadena summers. The children were a very lively bunch, and had a tendency (at least Linda and Peter did) to cruise through Crellin Laboratory and invite people up for swimming parties when their parents were away. There was one occasion, I believe it was during or just after such a party, when the senior Paulings came walking up the drive. I could only say, “But , I thought you were in Europe!” I do not remember their response. I do remember, though, that it did not seem to matter to either Peter or Linda whether their parents were there or not. The Paulings must have been sorely tried now and then, for once Ava Helen spoke firmly through one of the windows, “Linda, can you get rid of those people, your father and I want to swim!” It was a very reasonable request: there were so many people that there was almost no room in the pool. We all left very quickly.

The pool played a memorable role in Matthew Meselson‘s life as well

I became a graduate student of Pauling’s by an accident that involved a swimming pool. One afternoon in the summer of 1953, Peter Pauling, Linus’ middle son, had some friends over for a swim. Linus came out of the house, dressed in a tie and a jacket, and peered down at me (I was in the water, not at all well-dressed) and he said “Well, Matt, what are you going to do next year?” (You’ve got to have this picture in your mind – I am all wet, largely naked, looking up at the world’s greatest chemist, who is wearing a tie and a jacket. For someone growing up in California, that alone is intimidating). So I said I was going to go back to the University of Chicago, where I had been before, and I was going to be a graduate student of the Committee on Mathematical Biophysics. It was the only time I ever saw Linus looking amazed. After a moment he said, “But Matt, that is a lot of … baloney! Why don’t you come to Caltech and be my graduate student?” So I looked up at him and said, “Okay, I will.” And that was that.

Later on, the Paulings added a large pool adjacent to the “Big House” at Deer Flat Ranch, which is pictured below.

We’ll never know exactly when it started, but one thing is clear: be it for exercise, recreation or simple cooling off, swimming was very much ingrained into the fabric of life for Pauling and his family.

Illustration from "Linus Pauling: Vim, Vigor and Vitamins", Discover, November 1982. Photo by Joe McNally.

Illustration from “Linus Pauling: Vim, Vigor and Vitamins”, Discover, November 1982. Photo by Joe McNally.

Zia Mian Lecture Now Available Online

mian-graphic

The fully transcribed video of Dr. Zia Mian’s lecture, “Out of the Nuclear Shadow: Scientists and the Struggle Against the Bomb,” is now available on the website of the Oregon State University Libraries Special Collections & Archives Research Center.  Mian gave the talk on the occasion of his receipt of the Linus Pauling Legacy Award, presented on April 21, 2014.  Mian was the eighth recipient of this award, granted every other year by the OSU Libraries.

In his lecture, Mian provides an overview of the responsibilities that scientists have historically assumed with respect to nuclear issues, pointing to Linus Pauling and Leó Szilárd as particularly impactful examples for later generations. Moving to contemporary affairs, Mian paints a downbeat picture of current trends in the nuclear realm, noting the United States’ plan to massively modernize its nuclear complex and the continuation of sabre-rattling in nuclear-armed India and Pakistan.

In the midst of this alarming scene, Mian notes that the world’s attention is increasingly moving away from nuclear issues as climate change and other problems of the day capture the news cycle. Mian reiterates the devastating impact that a nuclear conflagration would make upon Earth; worldwide famine and extreme planetary cooling being among the likely outcomes. The scenario is such that Mian, in echoing the Pugwash Conference of 1955, suggests that “those who know the most are the most gloomy.”

Zia Mian directs the Project on Peace and Security in South Asia, at the Program on Science and Global Security. The editor of numerous books, his research and teaching focuses on nuclear weapons and nuclear energy policy, especially in Pakistan and India, and on issues of nuclear disarmament and peace. He has also produced two documentary films, “Pakistan and India Under the Nuclear Shadow” (2001) and “Crossing the Lines: Kashmir, Pakistan, India” (2004). He is Co-Editor of Science & Global Security, an international journal of technical analysis for arms control, disarmament and nonproliferation policy. He is also a member of the International Panel on Fissile Materials (IPFM).

Previously, he has taught at Yale University and Quaid-i-Azam University, Islamabad, and worked at the Union of Concerned Scientists, Cambridge (Mass.), and the Sustainable Development Policy Institute, Islamabad. He has a Ph. D. in physics from the University of Newcastle upon Tyne.

Our past coverage of Mian’s work and visit – including an exclusive interview conducted by the Pauling Blog – is available here.  Additional information on the history of the Pauling Legacy Award, as well as links to four additional past lectures by Roald Hoffmann, Roger Kornberg, Roderick MacKinnon and John D. Roberts, is available at the award’s homepage.

Roger J. Williams and the Continuing Quest for Good Health

williams-smiling

[Part 2 of 2]

Though ostensibly retired in 1971, the nutrition scientist Roger J. Williams continued to pursue numerous research and public advocacy interests with terrific energy. Among his many other activities, Williams was the only non-physician member of President Nixon’s Advisory Panel on Heart Disease, convened in 1972. In a letter to the Secretary of Health, Education, and Welfare, Williams showed that he was ahead of his time in recognizing the importance of nutrition in reducing the risk of cardiovascular disease and other maladies.

While no one knows why heart disease is so prevalent, it is highly probable that a primary cause lies in the fact that in our industrialized age the public chooses its food only on the basis of appearance and taste, and has not been educated to choose on the basis of nutritional value. Much of our food is processed, transported long distances and kept a long time, and the purveyors of food cater to those who want attractive and tasty foods and who pay little attention to its nutritional efficacy. Modern scientific prowess has not been utilized as it should have been. Nutritional science has lagged.

Of the nineteen other members of the committee, all M.D.’s, Williams guessed that a third knew nothing about nutrition.

To combat these trends, Williams called for the establishment of nutritional research centers and the promotion of nutritional science to medical doctors and the general public. The important points that Williams made in his letter underline many of the public health goals that the U.S. government is working towards today.

Williams evinced continued frustration with this issue in a letter sent to Linus Pauling in 1975. In it, he wrote

There are so many times when I would like to consult you on matters of mutual interest….My colleagues and I are engaged in a serious campaign to contribute to the improvement of the attitudes of medical scientists, including biochemists, toward nutritional science. We are convinced, as I know you are, that unrecognized nutritional considerations are embedded in a host of health-related problems and that they go unnoticed because of the cavalier attitude toward nutrition generally adopted by medical schools and medical scientists.

In support of his continuing efforts to evoke change within the medical system’s view and understanding of nutrition, Williams edited the Physician’s Handbook of Nutritional Science, published in June 1975.  Two pieces written by Pauling – one on vitamin E and another on orthomolecular theory – were included in the text. Pauling also wrote a review of the book which, perhaps unsurprisingly, was largely favorable.

This book on nutritional science, written for physicians, may help the physicians of the United States to make up for a serious deficiency in their training…. [It] presents a generally sound treatment of nutritional science, which almost every physician could benefit by reading.

Pauling’s main criticism of the handbook was its (in his view) overly conservative treatment of vitamin C recommendations. Williams’ book suggested doses of 250 milligrams up to 1 gram of vitamin C per day; meanwhile Pauling was advocating intake as high as 10 grams per day for treatment of some diseases. This quibble aside, Pauling praised Williams’ information on biochemical individuality and the diverse nutritional needs of individuals. He also cheered the text’s sections on on orthomolecular psychiatry.


Roger Williams with the Paulings, ca. 1970s.

Roger Williams with the Paulings, ca. 1970s.

In 1976, Pauling and Williams established the Foundation for Nutritional Advancement. This non-profit organization was created to support research in the field of nutrition and to integrate nutrition into the study and practice of medicine – all areas that Williams and Pauling felt to be mostly neglected in the scientific world. The foundation issued grants to a diverse range of nutrition researchers, studying topics ranging from mental health to a wide variety of chronic diseases. It also hosted international conferences on nutrition, including one in Japan and one in China.

In announcing the launch of the organization, Williams wrote an open letter describing his and his colleagues’ motivations and intentions:

We have become increasingly and alarmingly concerned in recent years that a major aspect of health care is being almost totally neglected by medical schools and by practicing physicians….This major phase of health care which is sadly neglected involves monitoring the internal environments which surround the cells and tissues of our bodies and brains. We monitor the air and water as a matter of course, but who looks after the approximately 40 nutrient chemicals which come from the outside world and enter into our inner environments and make life and health possible? The answer is essentially, ‘No one.’

The open letter was also released to coincide with the publication of Williams’ The Wonderful World Within You: Your Inner Nutritional Environment, a book that covered the important nutritional information that Williams thought necessary for maintaining one’s health. As with Pauling’s later How to Live Longer and Feel Better, the book was written for a broad audience: Williams hoped that it would gain traction with the general public and find its way into high school curricula.

The publication of The Wonderful World Within You came about at the same time that Williams was fighting his own personal battle with mouth cancer, which was treated with chemotherapy and surgery. Although he did not use ascorbic acid as part of his own regimen, Williams’ experience convinced him that ascorbic acid should be further studied for use in cancer treatment.

As the years went by, Pauling and Williams stayed connected. In November 1979, Williams was honored by the Foundation for Nutritional Advancement at the University of Texas-Austin, with Pauling delivering the keynote lecture, focusing on his vitamin C research. The two also continued to correspond about articles that they were working on. Pauling provided considered feedback on several of Williams’ drafts, lauding him for many of the points that he made, though continually disagreeing about the place of religion in science. (Pauling made several attempts to persuade Williams to look into the Unitarian Universalist Church.) Pauling also nominated Williams for a number of awards, including the Nobel Prize, a decoration that, alas, eluded Williams.

williams-portrait

Williams did not fully retire until 1986 at the age of 92. In total he wrote twenty-one books – including several widely used organic chemistry and biochemistry textbooks – and nearly 300 scientific articles. He was a member of the National Academy of Sciences and, in 1957, served for a year as President of the American Chemical Society. He also received honorary doctorates from Columbia University, Oregon State University, his former place of employment, and the University of Redlands, his undergraduate alma mater.

Roger John Williams died of pneumonia on February 20, 1988 at the age of 94. When Pauling learned of Williams’ passing he sent a letter of sympathy and admiration to Roger’s wife, Phyllis. “Roger was a great man,” he wrote. “It has been one of my pleasures during recent years to have been closely associated with him. It is now about 58 years since I met him, in Corvallis. He was full of enthusiasm about his work on vitamins.”

Roger J. Williams: Nutrition Scientist

Roger J. Williams and Linus Pauling, 1972.

Roger J. Williams and Linus Pauling, 1972.

[Part 1 of 2]

“For about 15 years I have been working in the field of nutrition and I’ve become acquainted with many of the nutritionists, professors of nutrition. I have formed the opinion that Professor Williams is the outstanding man in this field in the world. I think that he has had the better background of training in the basic sciences which has permitted him to attack problems in this field more effectively than any other person.”

-Linus Pauling, November 1979.

Roger John Williams was a prolific scientist in the fields of biochemistry and nutrition who discovered pantothenic acid (vitamin B5) and named and researched folic acid (vitamin B9). He was also an important advocate of public health nutrition. In his writings, Williams emphasized the biochemical diversity of humans and the importance of studying individuals and their different internal environmental requirements through the prism of nutrition. As with Linus Pauling, a large part of Williams’ legacy is one of wide promotion of the importance of nutrition in health and preventative medicine.

Williams was born in Ootacamund, India, to U.S. Baptist missionary parents, on August 14th, 1893. His family returned stateside when he was two years old and he grew up in Kansas and California. He received his bachelor’s degree from the University of Redlands in 1914 and a high school teacher’s certificate from the University of California, Berkeley the following year. His undergraduate experiences with organic chemistry discouraged his initial inclinations toward graduate study in chemistry, and he chose instead to teach chemistry and physics at a local high school. During this time he also married Hazel Wood, his college sweetheart. They later raised three children together and were married for thirty-five years.

Roger Williams as a young man.

Roger Williams, age 16.

After two difficult years of teaching high school, Williams decided at last to pursue graduate school at the University of Chicago, the institution from which all three of his older brothers had graduated. Williams overcame his fear of organic chemistry with the help of a influential professor and earned his M.S. in 1918 and his Ph.D. one year later. His doctoral thesis was titled The Vitamin Requirement of Yeast, scholarship that attracted an unusual amount of attention and that proved to be the basis for much of his later work on nutrition.

Williams departed Chicago to become a professor at the University of Oregon, eventually moving to our own Oregon State University, then known as Oregon State College or OSC. During his two decades in Oregon, he continued to study yeast and human nutritional science, research that promoted the use of microorganisms such as yeast and bacteria in nutritional studies. The use of these substances sped up nutritional experimentation greatly and played an important role in advancing the fields of enzymology, genetics, and molecular biology.

While at OSC in 1933, Williams discovered and isolated pantothenic acid, also known as vitamin B5, an essential vitamin for synthesizing coenzyme-A and synthesizing and metabolizing proteins, carbohydrates, and fats. He later won both the Mead Johnson Award from the American Institute of Nutrition and the Chandler Medal from Columbia University for this discovery.

Not long after, in 1936 Williams’ oldest brother, Robert, synthesized and isolated aneurin (now called thiamin or vitamin B1), an important vitamin for human neurological processes. Roger Williams later discovered that thiamine is also important for yeast growth.


Williams during his graduate school days at the University of Chicago.

Williams during his graduate school days at the University of Chicago.

Williams and Linus Pauling met at Oregon State College, where Pauling had received his baccalaureate degree in 1922. In 1936 Williams and Pauling began to correspond about Williams’ research on pantothenic acid, Williams requesting Pauling’s help in determining the structure of the substance using x-ray crystallographic techniques. Pauling agreed to help because he was very interested in Williams’ research, and the two continued their correspondence into the following year.

Amidst this scientific collaboration, Williams also wrote to Pauling to complain about the state of the chemistry department at OSC. Pauling, in turn, wrote a letter to the state’s chancellor of higher education, suggesting that the head of the OSC chemistry department, Professor John Fulton, retire and be replaced by Roger Williams. Pauling wrote a glowing recommendation of Williams, noting that

Professor Williams is recognized throughout the country as an outstanding teacher of chemistry and an outstanding research man. His text-books in organic chemistry and biochemistry are widely used and show him to be a thoroughly well trained and able chemist and teacher. His researches and in particular his recent work on pantothenic acid constitute the most important chemical contribution that has been made from Oregon.

Pauling’s interest in the situation did not end with this recommendation. After a visit to Corvallis to give a speech for the Sigma Xi scientific research society, Pauling investigated Fulton by writing a letter of inquiry to Harvard University. He found that Fulton had only finished one course at Harvard, for which he received a C. The rest of his coursework had never been completed. Williams and Pauling thus concluded that Fulton had a phony master’s degree on his vita.

Pauling’s advocacy of Williams apparently fell on deaf ears. In December 1939 Williams wrote to Pauling of a deteriorating environment at OSC and his decision to move on.

I have come to the decision that I must sever my connection with this institution as soon as I can make arrangements to locate elsewhere….The atmosphere in which I have found myself has often not been stimulating and continual annoyances are bound to wear away one’s spirit.

Williams’ departure was Oregon State’s loss; as it turned out, Pauling was correct in his evaluation of Williams’ abilities.


The decision to move having been made, Pauling continued to look out for Williams’ interest, writing query letters to multiple universities recommending the addition of Williams to their departments. In short order, Williams found a position as professor at the University of Texas at Austin. Williams expressed gratitude to Pauling for his assistance in the process and the two made a habit of sharing ideas on possible additions to each other’s departments for many years.

Williams ca. 1950s.

Williams ca. 1950s.

In 1941 Williams founded the Clayton Foundation Biochemical Institute at the University of Texas, serving as its director until 1963. Under Williams’ leadership, more vitamins and their variants were discovered at the Clayton Institute than at any other laboratory in the world. It was during this period that Williams first concentrated and named folic acid, or vitamin B9, an essential vitamin for DNA processes and red blood cell production. Sadly, it was also during this period, in 1952, that Williams’ first wife Hazel died. He married Mabel Phyllis Hobson the next year and the couple traveled extensively together all over the world, remaining happily married until Roger’s death in 1988.


In 1964 the volume of letters exchanged between Williams and Pauling began to increase, because Williams was writing a book and he wanted Pauling’s input. You Are Extraordinary, published in 1967, emphasizes as its central theme the crucial need for scientists to consider people as individuals, rather than focusing on the average human being. Pauling respected this idea so much that he devoted a whole chapter of his own book, Vitamin C and the Common Cold, to Williams’ ideas, extrapolating from them that individuals have unique vitamin C requirements, person to person.

Williams later in life.

Williams later in life.

In 1970 Williams made news through his publication of an article about an experiment that he conducted on rats in which he fed standard enriched white bread to one group and bread further enriched with trace minerals, vitamins, and protein to a second group. The second group fared much better than the first and he used these results to argue that bread manufacturers in the U.S. should change their enrichment protocols to add more nutrients. In response, corporations in the bread industry stated that they would not make any changes until they were recommended by the Food and Drug Administration.

Interestingly, Williams’ older brother Robert was the scientist who devised the original enrichment recommendations. Enrichment standards are necessary because the typical industrial process of milling white flour in the U.S. removes many of the important nutrients naturally available in grains. Before white bread was enriched, many Americans suffered from B vitamin deficiencies. Roger Williams argued that his brother’s original recommendations were good in 1941, but that thirty years later they could be markedly improved upon.

Williams’ push coincided with problems that Linus Pauling had been facing in his own nutritional research. Both scientists felt that nutrition research was not well respected by medical doctors and most scientists, and thus its importance was downplayed or disregarded. Because of the low degree of institutional esteem afforded to work on nutrition, insufficient funding was available to the field.

Though fighting headwinds on numerous fronts, Roger Williams was well-respected within his own community of researchers.  In alignment with Pauling’s ideas related to orthomolecular psychiatry, he served as a founding fellow of the Academy of Orthomolecular Psychiatry in 1971. That same year, Williams became an Emeritus Professor of Chemistry at the University of Texas, though as we’ll see, the vigor of his work did not diminish in retirement.

Think Independently: Pauling’s Years at OAC

 

Linus Pauling on OAC graduation day, June 1922.

Linus Pauling on OAC graduation day, June 1922.

[Ed Note: Last Saturday, Oregon State University graduated its largest class ever.  In honor of the class of 2014, we're taking a quick look back at Linus Pauling's years as an undergraduate at what was then known as Oregon Agricultural College.]

It might be said that brilliant ideas start with reflections on problems of daily life. The undergraduate story of young Linus Pauling traces the growth of a remarkable talent emerging from an interest in tackling the familiar problems of ordinary life. Pauling enrolled in Oregon Agriculture College (OAC) at the young age of 16 in the Fall of 1917 and graduated from OAC with a B.S. in chemical engineering in June 1922. In those five years, he matured in a significant way, both socially and academically. In school, he not only earned A’s in all of his chemistry and mathematics courses, he also stoked his passion for science and even began to approach the burgeoning field of physical chemistry – a landscape of study that he would one day play a major part in defining.

At OAC and in the decades that followed, a main propellant of his growth and success was Pauling’s persistence in thinking independently. Years later, Pauling famously suggested to a crowd of young people that, “When an old and distinguished person speaks to you, listen to him carefully and with respect – but do not believe him. Never put your trust into anything but your own intellect.”  The kernel of that idea was apparent during young Pauling’s stint in Corvallis.

Pauling at the Oregon coast with his cousin Rowena, 1918.

Pauling at the Oregon coast with his cousin Rowena, 1918.

One quality shared by most successful scientists is that they love and excel at thinking. At an early age, Pauling started to show the signs of an independent thinker, always seeking to dig deeper into a question once he was drawn to it. Pauling’s grandparents lived in Oswego, Oregon close to the newly built Portland Cement Company. On weekend visits to his grandparents, Pauling, aged 14, frequently went to the cement plant’s laboratory and spent hours there bombarding the chief chemist with questions. Many years later, Pauling remembered this patient individual as “a man who was not very interested in chemistry, but who served as scoutmaster and who was willing to talk with me and to answer my questions.”

Pauling continued to pursue this interest during his college years. In the summer of 1919, mostly due to his need to earn money to pay for school but likely also motivated by his early contacts with the cement industry, Pauling secured a summer job in southern Oregon as a blacktop pavement inspector. His main task was to monitor the quality of the bitumen-stone mixes comprising the pavement. In June 1920, these activities were crystallized in his first scientific publication, “The Manufacture of Cement in Oregon,”  which appeared in The Student Engineer. In his three-page article, Pauling specified the process by which cement was produced, from crushers cutting large rocks as a first step to the kilns yielding the final, small round particles for cooling in the finishing mill.

Freshman fun at OAC, 1918. Photo by Linus Pauling.

Freshman fun at OAC, 1918. Photo by Linus Pauling.

Pauling’s digging into the cement industry was an indication of his ability to think independently and pursue a problem persistently, a set of traits that informed his academic work as well. His course reports from the metallography lab in the spring of 1921 also provide a nice glimpse into his scientific acumen and his growing confidence. The reports are not written in the formal and impersonal manner that one might to expect to find. Instead, quite often, Pauling used plain terms and interjected many of his own thoughts in the write-ups. From item to item, a personal voice is easily identified and the reports make for engaging reading. One interesting example is the concluding paragraph to Pauling’s report on “Preparation and Examination of Specimens” (April 25, 1921), which is typically lighthearted and even boastful.  Presumably addressing his professor, Pauling writes

I have made free use of technical terms throughout on the assumption that you would understand them, but in case you do not, I refer you to my experiment on metallography the first quarter of this year, in which complete definitions are given. I have also attempted to use words of one syllable to as great an extent as is practicable in order to prevent any mental strain. Let me repeat that, for a really good article, you should read my previous experiment.

When approaching a lab topic, Pauling early on developed the habit of consulting all of the relevant literature that he could find in developing a general picture of the status of current research on that topic. In his reports, he often gave his opinions of the literature in a very frank manner. See, for example, this aside included in a 1921 report titled “Heat Treatment and Tests of Specimen and Case-Carburizing.”

Quite often in reading, I wonder where people find all the things they do to write about. Just about as often I wonder what the idea of writing so much is, and why it is necessary to really do it. Then again I find the secret. It is this. All this writing is necessary because we are acquiring so much knowledge that we are behind in writing it down as it is, and there is still room for more books. I wish that someone would prepare (or rather, had already prepared) a short concise article on heat treating steels covering about five such pages as this. His work at least would be considered useful by me.

While sharpening his scientific toolkit at the theoretical level, the undergraduate Pauling also loved the mathematical rigor required by many technical tasks. In pursuing these tasks, he developed a stickler’s personality, one that reveals itself time and again in his correspondence over the years.

One such early example is dated March 15, 1920, in a letter from Pauling to Dr. George Smith, the author of a chemistry textbook.  In it, Pauling – who had just turned 19 years old – points out a tiny technical error. On page 11 of the textbook, Smith talked about errors in weight measurement. Using the tools of the day, Smith pointed out that weighing the same sample of a given substance twice would always yield two results which were very slightly different, meaning that there is always a built-in percentage error in weight measurements.

Smith went on to explain how observers calculated errors of this sort in weighing mixtures, using as an example the measurement of the weight of a sample clay which contained in it 0.2% of magnesium oxide. Here Pauling found a point of disagreement; he thought the calculation of the percentage error of the magnesium oxide was problematic. In his text, Smith stated that if the measuring error was 0.1% in total, then for the impurity in the sample, which was 0.2% of the total clay, the percentage error would be 0.0002%. Conversely, Pauling thought the error in the weight of the impurity should be compared with the impurity itself, and that the percentage error would thus be 0.1%, instead of 0.0002%.

Ready to take on the world. 1922.

Ready to take on the world. 1922.

A few days later, on March 22, Smith wrote back to his young correspondent in a very pleasant tone, saying that although he did not agree that this was an error, he admitted that the example was confusing and  needed further clarification. What’s more, at the end of the letter, Smith enthusiastically mentioned that he was looking forward to meeting Pauling at the upcoming meeting of the Pacific Division of the American Association for the Advancement of Science.

Before and during his years at OAC, Pauling thought independently about scientific problems, making every effort to find answers that were satisfying to him. He doggedly queried expert opinions, widely searched the available literature, and critically judged the information that he gathered. Only then would he put forward and test his own proposals. He was interested in industrial problems and theoretical questions alike. He was engaged with topics in the lab and gradually developed an acute scientific insight.  And by the time he left Oregon Agricultural College, he was well-poised to do great things as a scientist.

Pauling and Perutz: The Later Years

[Concluding our series on Max Perutz, in commemoration of the Perutz centenary.]

In 1957, Max Perutz and Linus Pauling wrote to each other again on a topic that was new to their correspondence. This time Pauling asked Perutz to sign his petition to stop nuclear weapons tests, a request to which Perutz agreed.

Signature of Max Perutz added to the United Nations Bomb Test Petition, 1957.

Signature of Max Perutz added to the United Nations Bomb Test Petition, 1957.

As the decade moved forward, the discovery of the double helical structure of DNA attracted ever more attention to the work of James Watson and Francis Crick. In May 1958, Perutz asked Pauling to sign a certificate nominating his colleagues Crick and John Kendrew to the Royal Society. Pauling agreed, though stipulated that Kendrew’s name be placed first on the nomination, as he expected that Crick would get more support. As with Pauling’s bomb test petition a year earlier, Perutz agreed.

At the beginning of 1960, William Lawrence Bragg wrote to Pauling about nominating Perutz, along with Kendrew, for the 1961 Nobel Prize in Physics. Pauling was hesitant about the nomination, thinking it was still early, as their work on hemoglobin structure had only recently been published. Pauling also felt that Dorothy Hodgkin should be included for her work in protein crystallography. Bragg thought this a good idea and included Hodgkin in his nomination.

By March, Bragg’s nominations had gone through and Pauling was asked to supply his opinion. After spending some time thinking about the matter, Pauling wrote to the Nobel Committee that he thought that Robert B. Corey, who worked in Pauling’s lab, should be nominated along with Perutz and Kendrew for the Nobel Prize in Chemistry instead. Pauling felt that if Perutz and Kendrew were included in the award, Corey should be awarded half, with the other half being split between Perutz and Kendrew. Pauling also sent a letter to the Nobel Committee for Physics, indicating that he thought that Hodgkin, Perutz, and Kendrew should be nominated for the chemistry prize. Pauling sent a copy of this letter to Bragg as well.

Pauling’s letter to the Nobel Committee, March 15, 1960. pg. 1.

Pg. 2

In July, Bragg replied to Pauling that he was in a “quandary” about Corey, as he was “convinced that” Corey’s work “does not rank in the same category with that which Mrs. Hodgkin or Perutz and Kendrew have done.” Perutz and Kendrew’s efforts, he explained, had theoretical implications directly supporting Pauling’s own work, whereas Corey’s research was not that “different from other careful analyses of organic compounds.” Once everything was sorted out, Perutz and Kendrew were awarded the Nobel Prize for Chemistry in 1962 (the same year that Watson and Crick, along with Maurice Wilkins, won in Physiology/Medicine, and Pauling, though belated for a year, won the Nobel Peace Prize) and Hodgkin received the Nobel Prize for Chemistry in 1964. Robert Corey never was awarded a Nobel Prize.


Linus Pauling, Max Delbrück and Max Perutz at the American Chemical Society centennial meeting, New York. April 6, 1976.

Perutz and Pauling corresponded very little during the 1960s, with Perutz writing only to ask for Pauling’s signature, once for a photograph that would be displayed in his lab and a second time for a letter to Italian President Antonio Segri in support of scientists Domenico Marotta and Giordano Giacomello, who were under fire for suspected misuse of funds.

In 1971 Perutz read an interview with Pauling in the New Scientist which compelled him to engage Pauling on scientific questions once again. Perutz was surprised to have read that Pauling had tried to solve the structure of alpha keratin as early as 1937 and that his failure to do so led him to study amino acids. Perutz wrote that had he known this in 1950, he, Bragg and Kendrew might not have pursued their own inquiry into alpha keratin. Pauling responded that he thought his efforts had been well-known as he and Corey had made mention of them in several papers at the time. Pauling explained that he had difficulties with alpha keratin up until 1950, when he finally was able to show that the alpha helix best described its structure. Perutz replied that he was aware of Pauling and Corey’s work and the alpha helix, but was surprised that Pauling’s early failure to construct a model led him to a more systematic and fruitful line of research.

Perutz also wondered whether Pauling had seen his article in the previous New Scientist, which reflected on Pauling and Charles Coryell’s discovery of the effect of oxygenation on the magnetic qualities of hemoglobin. Perutz saw this as providing “the key to the understanding of the mechanism of haem-haem [heme-heme] interactions in haemoglobin.” Pauling responded that he had not seen Perutz’s article but would look for it, and also sent Perutz a 1951 paper on the topic. Perutz took it upon himself to send Pauling his own article from the New Scientist.

A few years later, in 1976, Perutz again headed to southern California to attend a celebration for Pauling’s 75th birthday, at which he nervously gave the after dinner speech to a gathering of 250 guests. Before going to the event in Santa Barbara, Perutz stopped in Riverside and visited the young university there, which impressed him. Perutz wrote to his family back in Cambridge that he wished that “Oxbridge college architects would come here to learn – but probably they wouldn’t notice the difference between their clumsy buildings and these graceful constructions.”

Perutz also visited the Paulings’ home outside Pasadena, which elicited more architectural comments. Perutz described to his family how the Pauling house was shaped like an amide group, “the wings being set at the exact angles of the chemical bonds that allowed him to predict the structure of the α-helix.” Perutz asked Pauling, perhaps tongue in cheek as he thought the design somewhat conceited, “why he missed the accompanying change in radius of the iron atom.” Pauling replied that he had not thought of it.

Bertrand Russell and Linus Pauling, London England. 1953.

In preparation for his speech, Perutz also took some time to read No More War! which he concluded was as relevant in 1976 as when it was first published in 1958. Perutz saw Pauling’s faith in human reason as reminiscent of Bertrand Russell’s. Indeed, the many similarities between the two were striking to Perutz, and he included many of them in his talk, “except for their common vanity which I discreetly omitted.” In a personal conversation, Perutz asked Pauling about his relationship with Russell which, as it turned out, was mostly concerned with their mutual actions against nuclear weapons. Perutz was somewhat disappointed that “they hardly touched upon the fundamental outlook which I believe they shared.”


Perutz and Pauling were again out of touch for several years until April 1987, when Pauling traveled to London to give a lecture at Imperial College as part of a centenary conference in honor of Erwin Schrödinger. Pauling’s contribution discussed his own work on antigen-antibody complexes during the 1930s and 1940s, during which he shared a drawing that he had made at the time. Perutz was in attendance and noticed how similar Pauling’s drawing was to then-recent models of the structure that had been borne out of contemporary x-ray crystallography. Perutz sent Pauling some slides so that he could judge the similarities for himself.

Flyer for Pauling's 90th birthday tribute, California Institute of Technology, February 28, 1991.

Flyer for Pauling’s 90th birthday tribute, California Institute of Technology, February 28, 1991.

The final time that Pauling and Perutz met in person was for Pauling’s ninetieth birthday celebration in 1991. Perutz, again, experienced stage fright as he gave his speech. But he was encouraged afterwards, especially after receiving a compliment from Francis Crick who, according to Perutz, was “not in the habit of paying compliments.” Perutz told his family that the nonagenarian Pauling “stole the show” by giving one speech at 9:00 AM on early work in crystallography and then another speech at 10:00 PM on his early years at Caltech. Perutz found it enviable that Pauling stood for both lectures and was still getting around very well, though he held on to the arm of those with whom he walked. Without coordinating, Perutz and Pauling also found a point of agreement in their talks, noting that current crystallographers were “so busy determining structures at the double” that they “have no time to think about them.” This rush often caused them to miss the most important aspects of the newly uncovered structures.

Just as Perutz first encountered Pauling through one of his books, The Nature of the Chemical Bond, so too would Pauling’s last encounter with Perutz be through a book, Perutz’s Is Science Necessary? Pauling received the volume in 1991 as a gift from his friends and colleagues Emile and Jane Zuckerkandl. Pauling’s limited marginalia reveal his interest in the text’s discussions of cancer and aging research. Aged 90 and facing his own cancer diagnosis, Pauling was particularly drawn to Perutz’s review of François Jacob’s The Possible and the Actual which sought, but did not find, a “death mechanism” in spawning salmon. Pauling likewise highlighted the book’s suggestion that “like other scientific fantasies…the Fountain of Youth probably does not belong to the world of the possible.” And Pauling made note of particular individuals that he had known well, like John D. Bernal and David Harker. Pauling deciphered the latter’s identity from Perutz’s less-than-favorable anonymous portrayal.

Pauling also noted spots where Perutz wrote about him. While most of these references were positive and focused on topics like Pauling’s influence on Watson and Crick and his breakthroughs on protein structure, one in particular was not. Perhaps less cryptic than the reference to Harker, Perutz described how “one great American chemist now believes that massive doses of vitamin C prolong the lives of cancer patients,” following it with “even more dangerous are physicians who believe in cancer cures.”

While critical, Perutz really meant the “great” in his comment and he continued to repeat it elsewhere. After Pauling passed away in August 1994, Perutz told his sister Lotte that “many feel that he [Pauling] was the greatest chemist of this century” while also being “instrumental in the protests that led to Kennedy and Macmillan’s conclusion of Atmospheric Test ban.”  He reiterated this idea in the paragraph that concluded his obituary of Pauling, published in the October 1994 issue of Structural Biology.

Pauling’s fundamental contributions to chemistry cover a tremendous range, and their influence on generations of young chemists was enormous.  In the years between 1930 and 1940 he helped to transform chemistry from a largely phenomenological subject to one based firmly on structure and quantum mechanical principles.  In later years the valence bond and resonance theories which formed the theoretical backbone of Paulings work were supplemented by R. S. Mullikens’ molecular orbital theory, which provided a deeper understanding of chemical bonding….Nevertheless resonance and hybridization have remained part of the everyday vocabulary of chemists and are still used, for example, to explain the planarity of the peptide bond.  Many of us regard Pauling as the greatest chemist of the century.

Follow

Get every new post delivered to your Inbox.

Join 56 other followers