Remembering Henry Taube

1983i.131

Four Nobel Prize-winning chemists with a connection to Stanford University. From left, Arthur Kornberg, Paul Flory, Henry Taube, and Linus Pauling. This photo was taken in 1983 on the day that Taube received notification of his having been awarded the Nobel Prize in Chemistry.

[Ed Note: This is our final post for 2015.  Thanks for reading and please check back in early January for more!]

This month marks the 32nd anniversary of Henry Taube’s Nobel Prize in chemistry, awarded for ‘Electron Transfer between Metal Complexes.’ His seminal paper on the subject was 30 years old when he received the Nobel Prize, but the correlation that he described in it remained the predominant theory at the time of his receipt of the Nobel medal. Taube would have turned 100 this past November 30th , 2015. He died in 2005 in his home in Palo Alto, California at 89 years old.

Linus Pauling, for many years a friend of Taube, wrote to him in 1983 to congratulate him on his prize, calling it a “fine honor.” Pauling also kept a newspaper clipping announcing Taube’s Nobel in a collection of his personal memorabilia. In it, Taube attributed his success in Stockholm to a “domino theory” of scientific awards: once they started coming, you just seemed to get more of them. “I have to pay for it by giving a speech,” Taube said.  And indeed, Taube received numerous other decorations, including the Priestley Medal in 1985.


Born in the small town of Neudorf, Saskatchewan, Henry Taube was the youngest of four boys. The son of German immigrants who moved from the Ukraine and settled in Canada in 1911, Taube reflected fondly on his experiences growing up, noting

Certainly, there is nothing about my first 21 years in Saskatchewan, taken in the context of those times, that I would wish to be changed. The advantages that I enjoyed include: the marvelous experience of growing up on a farm, which taught me an appreciation of nature, and taught me also to discipline myself to get necessary jobs done.

Two years after the completion of his PhD at UC Berkeley in 1940, Taube became a naturalized citizen of the United States. As a young academic, he began studying the chemistry and photochemistry of non-metallic oxidants such as ozone, hydrogen peroxide, and halogens, and their reactions with a variety of inorganic and organic substances. Taube also worked on the subject of electron transfer in chemical reactions for most of his professional life, stating in his Nobel lecture that,

by an accident of history, I was a graduate student at the University of California, Berkeley, about the time the first natal stirrings of [this] subject occurred, and at a place where those stirring were most active.

His interest in the measurement of the rates of self-exchange reactions was shared by many, but not reflected in research or development for years to come. Students who might have harbored plans to carry out such experiments, Taube later pointed out, became engaged in war-related activities instead.

Taube’s first academic appointment was as an assistant professor at Cornell, where he engaged in the study of oxidation-reduction reactions, or redox reactions. In 1943 he began his correspondence with Linus Pauling, asking him to visit Cornell and deliver a lecture on antibody reactions, one of Pauling’s areas of specialty at the time. Pauling declined, stating that he would not be traveling in the vicinity of Ithaca any time soon. Taube tried again to meet with Pauling while on a trip to UCLA in 1949, but Pauling was out of his office.

It is something of an irony that Taube, anxious to connect with such an eminent figure in chemistry, would become the chair of a department where Pauling would work later in life. While the pair did not have much luck connecting in the 1940s, forty years later they would regard one another as close companions.


Taube_sitting

As an associate professor at the University of Chicago, Taube studied charge transfer complexes, describing metal-ligand bonds in terms of molecular orbital language. As a result, the new field of mixed-valence compounds began to develop. Taube’s continued study in this area united the divergent disciplines of classical coordination chemistry and organometallic chemistry, bringing inorganic chemistry into a more modern age.

Taube’s contributions were notable as confusion between thermodynamic and kinetic stability of coordination compounds had plagued coordination chemistry for decades, hindering theoretical advancement in the field. Classical coordination chemistry was created by Alfred Werner in 1893, with little groundbreaking work in the area come to pass in the four decades following. At this same time, organic and biological chemistry were progressing in exciting ways, in no small part due to work being conducted by Linus Pauling. Indeed, in organic chemistry, Pauling’s influence is ubiquitous: the mechanisms of organic substitution reactions, the discovery of biochemical cycles and molecular disease, the role of vitamins and antibiotics – all were touched by his genius. But for inorganic chemistry, even Pauling’s valence bond theory did not prompt advancement. This all began to change with Henry Taube.

By shifting focus from classical coordination chemistry toward the mechanisms of redox reactions, Taube affected an important shift that revitalized inorganic chemistry. Specifically, Taube established a dichotomy between inert and labile complexes, using valence bond theory to frame the definitions of these metal ions. The effect on inorganic chemistry was so monumental, it has since been dubbed by some as the “Taube Revolution.” Published in 1952, Taube’s “Rates and Mechanism of Substitution Reactions in Inorganic Complexes in Solution” is a foundational work. This was an important personal year for Taube as well; it was the year that he married his wife, Mary. They would have four children; Karl, Heinrich, Linda and Marianna.


By the early 1970s, Taube was chairman of the Chemistry Department at Stanford University, where Pauling too was a faculty member. When Pauling was reclassified as an emeritus member of the faculty in 1972, a memo from Taube to Calvin Quate, the associate dean of humanities and sciences at Stanford, made his opinion of Pauling’s situation clear: “Linus Pauling’s contributions to our department are much valued,” Taube clarified for Quate. “It is the intention of the Executive Committee to recommend reappointment on a year-by-year basis for as long as he continues to be effective in supervising a research program.”

The following year, Pauling wrote to Taube to express concern about his position. In his response, Taube pointed out that, though now classified as a professor emeritus, the administration’s action did not change Pauling’s current appointment as regular faculty, which would remain in force until 1974. After that time, as indicated by Taube in his memo to Quate, Pauling would continue to be reappointed as long as he remained “productive in scientific work.” Taube added, “I feel confident that the change in nominal status next fall will not interfere with your scientific program.”

Over the years, the two men enjoyed a lively correspondence on many issues related to work and pleasure. Taube sent Pauling reprints of his papers, and asked Pauling just before receiving his Nobel Prize, “When you first formulated your ideas on back bonding, did you have any inkling of what its ramifications might be?” (in this, Taube was referring to his own work with redox reactions in metal complexes.) Taube added, “After things settle down, post-Stockholm, Mary and I hope to get together with you again socially.”

Taube also referred to Pauling as the living person whom he most admired, and the two saw eye to eye on many issues. In particular, Taube used his position as a Nobel laureate to argue for educational reform and nuclear disarmament, which he saw as the country’s most important issues in the 1980s. “I’m appalled not that the general public tends to be rather ignorant,” Taube explained, “but they don’t even care about the scientific issues.” All informed citizens, Taube thought, needed to know the basics, and in this he agreed with Pauling. “The training that science teachers get simply isn’t adequate for the job in the elementary schools,” he said. “The solution is to improve science teaching for teachers, and pay them a wage commensurate with their responsibilities.”


taube in lab

Though in many ways Taube is to inorganic chemistry what Pauling was to the organic side, Taube’s work has also been described as setting the stage for electron transfer studies in organic areas, including peptides, proteins, and other complex biomolecules –  all areas of study crucial to many of Pauling’s interests. This is presumably one reason why Pauling recruited Taube to support the Linus Pauling Institute of Science and Medicine.

The connection between Taube and the Institute began very early on, in 1972, when Pauling suggested to him that some of Taube’s graduate students might be interested in also working on orthomolecular studies with either himself or his assistant, Arthur B. Robinson. Twelve years later, in 1984, Pauling wrote to Taube asking him to join the Institute’s board of associates. Taube accepted, despite the fact that the Institute was involved in a very public battle with the Mayo Clinic, one based on what Pauling described in his letter as, “a thoroughly misleading account of [the Institute’s] work.”

In 1987 Pauling asked his friend to become even more involved, writing that he was pleased to tell him that the Board of Trustees had authorized him to ask Taube to join their rank and file. Taube accepted this position as well, but ultimately resigned in 1989, stating that he could “provide little help in solving the kind of [largely financial] problem that the Institute faces,” and that he believed he was “usurping an opportunity for service which others, of greater influence in financial or medico-scientific circles, could better fill.” Pauling was disappointed and disagreed with the decision, but responded simply that it would not otherwise impact Taube’s connection to the Institute.


Henry Taube’s love of chemistry and the impact that he made on the field seemed sometimes unbelievable to the man himself. Humble by nature, Taube offered in his Nobel lecture that he had only, “focused rather narrowly on electron transfer reactions between metal complexes.”

While Pauling and many others recognized and cited the importance of his work in developing a general principle of electron transfer, Taube remained much more cautious in his assessment. The principles that he had derived, Taube pointed out, manifested differently in different materials and reactions. Consequently, the descriptive chemistry of such relationships could be quite different.

Nonetheless, Taube saw these differing manifestations as an exciting challenge, describing them in his Nobel lecture as “the fabric of chemistry.”  In this love of scientific inquiry and the quest for a better understanding of the natural world, Taube was once again reunited with his close friend, Linus Pauling.

 

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An Interview with Balz Frei, Director of the Linus Pauling Institute

Balz Frei

Balz Frei

Oregon State University is turning 150 years old in 2018, and already several projects are being developed to mark the occasion.  One of them is a major oral history initiative that is capturing the stories of a wide array of alumni, faculty, staff, administrators and friends of OSU.

Several months ago, the project conducted an interview with Dr. Balz Frei, who has led OSU’s Linus Pauling Institute since 1997.  A Swiss native, Frei worked under Bruce Ames at UC-Berkeley before moving on to Harvard, the Boston University School of Medicine and, ultimately, Oregon State.

Frei’s research has always focused on the processes fundamental to human health. During his time in Berkeley, Frei became interested in vitamin C and met Linus Pauling. His later work has focused on oxidative stress and the role that it plays in atherosclerosis. He has also investigated arterial function and potential dietary compounds – including vitamin C – that might help prevent oxidation of LDL cholesterol.

Under Frei’s leadership, the Linus Pauling Institute has stabilized its funding base, hired several principal investigators and made substantial contributions to the published literature on subjects relating to nutrition and optimal human health.

In 2011 the Institute celebrated a major milestone with the completion of the Linus Pauling Science Center. This 105,000 square foot facility, built for $62.5 million, is the largest academic facility project in OSU history. Now housed in this new space, LPI continues to conduct research on cardiovascular and metabolic diseases, healthy aging, and cancer chemoprotection, and engages in public outreach through its Micronutrient Information Center and Healthy Youth Program.

Excerpts from Frei’s oral history interview, including his memories of meeting Pauling, his sense of Pauling’s vitamin C work, and his vision for the future of LPI, are included below the cut.

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The Discovery of Human Plastin at the Pauling Institute

Milestones in Plastin Research

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

In 1985 my lab at the Linus Pauling Institute of Science and Medicine (LPISM) in Palo Alto, California started to work on an abundant protein of white blood cells (lymphocytes, macrophages, etc.) that mysteriously appeared in human tumor-derived cells of solid tissues (carcinomas, fibrosarcomas, melanomas, etc). I had noticed this phenomenon a few years earlier while at the National Institutes of Health. I also noticed that this protein appeared in oncogenic virus-transformed (SV40 virus) human fibroblasts, but the protein was not expressed in the normal fibrolasts.

I was intrigued by the fact that a major protein of circulating blood cells would be induced during solid tumor cell development because it is well known that solid tumor cells become more anchorage-independent and can circulate like white blood cells to metastasize to other organs. My colleague, David Goldstein, took the lead in examining the expression of this mysterious protein in different cell types of fractionated white blood cells. At the time this protein was assigned only a number (p219/p220) corresponding to its position in two-dimensional protein profiles. We found that this protein was abundantly expressed in all normal white blood cell types that we examined but it was not expressed in normal cells of solid tissues (Goldstein et al, 1985).

When David’s paper was submitted to Cancer Research, the reviews came back positive and the paper was accepted for publication, but one reviewer asked that we give the protein a name. I was thrilled by the thought of naming a protein and its gene which would immortalize our work, so I took on the serious task of coming up with a name that had lasting meaning. My theory was that this cancer marker contributed in some then-unknown way to the plasticity of the cytoplasm in solid tumor cells because of its normal presence in circulating white blood cells. Also, I had seen the great movie, The Graduate, with Dustin Hoffman and recalled that amusing scene depicted in the picture included below. So I named the protein “plastin” – the greatest new thing since sliced bread. 🙂

The Graduate

That same year, I met Steve Kent from Caltech at a meeting in Heidelberg, Germany. After hearing my talk, Steve suggested that we collaborate. He mentioned that a postdoctoral fellow in Leroy Hood’s lab, Dr. Ruedi Aebersold, was trying to develop a more sensitive protein sequencing method for purposes of determining snippets of amino acid sequences from small amounts of unknown proteins eluted from two-dimensional gels (protein profiles) like the gels that we used to characterize plastin in David’s paper. If we could get an accurate partial sequence of plastin, we could devise a nucleic acid probe based on the genetic code that could be used to clone a plastin “copy DNA” from a cDNA library. If the plastin cDNA was cloned, we could then define the protein and perhaps its function by determining the nucleic acid coding sequence in the clone.

Madhu Varma.

Madhu Varma.

I gave Dr. Madhu Varma at LPISM the arduous task of isolating the plastin polypeptide “spot” for sequencing. Madhu cut out the stained spot from 140 two-dimensional gels, in effect purifying enough protein for sequencing by Ruedi at Caltech. Madhu succeeded and Ruedi produced eight short peptide sequences that could be used to develop short nucleic acid probes that would hybridize to the plastin cDNA clone isolated from a tumorigenic human fibroblast cDNA library.

Ching Lin.

Ching Lin.

Dr. Ching Lin at LPISM took one of the nucleic acid probes and immediately attempted to screen a tumorigenic fibroblast cDNA library. If we identified any clones that bound this probe, then we would perform a quick test to determine that we had cloned the plastin coding sequence. But science is full of surprises and we found that the first clone he isolated detected a gene product that was not in lymphocytes but only in normal human fibroblasts – in other words, it failed the test. This is where Ching’s brilliance took over. He was convinced that this first clone he had isolated was indeed a plastin coding sequence so he used this clonal DNA as a new probe against the tumorigenic fibroblast cDNA library. He isolated a new clone that passed the test and detected a gene that was expressed in lymphocytes and tumorigenic fibroblasts but not in normal human fibroblasts.

We performed other experiments that proved that we had cloned two different isoforms of plastin: L-plastin, expressed in lymphocytes and solid tumor-derived cells, and T-plastin that was expressed in normal solid tissues and co-expressed with L-plastin in tumor cells from solid tissues (Lin et al, 1988; Lin et al, 1990). Ultimately this work led to the complete characterization of the human plastin multigene family and verification that both isoforms were aberrantly expressed in various types of human tumors.

The figure at the top of this post maps the progression of discovery that followed our research, which began at the Pauling Institute in 1985. Our publications are shown in red in the graph and research published by other labs is shown in the blue bars.

Here are several plastin milestones discovered by other researchers:

  • T-plastin is abundantly induced in Sezary lymphomas, a lethal T-lymphocyte cancer (Su et al, 2003);
  • L-plastin induction in solid tumors contributes to invasive cancer growth and metastasis (Klemke et al, 2007);
  • Mutations in T-plastin play a role in the genetic disease Spinal Muscular Atrophy (Oprea et al, 2008); and
  • Most recently mutations in both L- and T-plastin promote re-growth of colon carcinomas following surgical resection of these tumors and chemotherapy (Ning et al, 2014).

These developments are more or less typical of the way science works. Progress in understanding complex phenomena like human cancer is the work of many scientists that builds on the observations of other scientists. This is just one example of the productive contributions in biomedical research that came about through early discovery research at LPISM in the 1980s.

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.

Some Personal Thoughts on Vitamin C in the 1980s and Now

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

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.

During my daily work for pharmaceutical and biotech clients, I am continuously learning about developments resulting from my research at the Linus Pauling Institute of Science and Medicine in Palo Alto, CA in the 1980s. Likewise, I am regularly coming into contact with new medically related developments focusing on vitamin C, an interest of Linus Pauling in those years.

With regard to our research on human plastins, a gene family of proteins that we discovered, cloned, and characterized at the Pauling Institute, it has recently been reported that plastin (PLS3) is a marker of carcinoma cells circulating in the blood (for example Yokobori, et al.). Our hypothesis was that when this protein was inappropriately expressed in cells from solid tissues, as it is in many tumor types, (e.g. carcinomas, fibrosarcomas, etc.) these potential tumor cells become more like blood cells in that they are able to live and replicate in an anchorage-independent state, an essential property of metastatic tumor cells. It is metastasis that kills us when we get cancer. Thus plastins, discovered and characterized at the Pauling Institute, may turn out to be the “holy grail” of cancer research.

I often run across new information on the medical importance of vitamin C without looking for it. Back in the 1980s, we would receive an annual shipment of loose vitamin C from Hoffmann-La Roche, Inc. as a way of saying thank you to Dr. Pauling for his advocacy of the merits of vitamin C. We received no funding from Hoffmann-La Roche though. One year I recall that two dignitaries from the company visited us. Dr. Pauling, with me and several others, walked our visitors to lunch a few blocks down El Camino Real in Palo Alto to my favorite restaurant, the Captain’s Cabin.

Afterward, while walking back to the Institute, one of the guests asked Dr. Pauling if he thought the perceived benefits of vitamin C were due to the placebo effect. I was amused because I too had said something ill-advised like that to Dr. Pauling in my first few months at the Institute. I mentioned to him that I had a vitamin C-resistant cold to which he replied, “You’re not taking enough!” and told me that he takes 18 grams a day. No doubt he had calculated this number based on the amount of vitamin C that animals produce within themselves every day. He would stir 18 grams into a large glass of water and imbibe the glass with no great rush.


A few months ago I heard a physician state in the national media that taking supplemental vitamins is a waste of money. This bold assertion reminded me of the announcement of the discovery of cold fusion and another premature announcement of the discovery of a cure for AIDS. The progress of science is slow but relentless, like the new developments with plastins fifteen years after I left LPISM’s labs.

On October 31, 2013, Kim, et al. at Seoul National University in South Korea published their findings on a new strain of experimental mice. The researchers knocked out the mouse gene encoding the enzyme L-gulono-γ-lactone oxidase, known as gulo for short. This is the gene that is missing in humans and that keeps us from synthesizing our own vitamin C, unlike nearly all other animals. An extreme lack of vitamin C in our diet can lead to scurvy, caused by aberrant expression of collagen in our connective tissues because of starvation of vitamin C in our diet. In these mice the lack of this gene caused “vitamin C insufficiency” in an animal model – a model that can now be used to learn more about the importance of vitamin C.

As these mice matured they expressed known blood markers of liver damage. This damage, called fibrosis, is basically the scarring of the liver, sort of like the scarring of the skin that is caused by certain types of skin damage. Concomitantly, as the mice aged, reactive oxygen species (ROS) and lipid peroxides increased in the liver, as did activated hepatic stellate cells, which deposited abnormal collagen fibriles on the basement membrane of functional liver cells. There is a wealth of evidence that elevated ROS in the lungs, liver, and kidneys is associated with pulmonary, hepatic, and renal fibrosis. Elevated vitamin C in these tissues will quench ROS.

Currently in the United States, there are no drugs approved to treat any of these forms of fibrosis. Fortunately, Intermune’s drug, pirfenidone, is close to approval for treatment of pulmonary fibrosis and has already been approved in Canada, Europe, and Japan. This drug reduces ROS and inhibits other key targets that are suspected of playing a role in the development of fibrosis. So who is to say that supplementing your diet with vitamin C is of no consequence? It is certainly not toxic in any way. Oh, by the way, pulmonary fibrosis is worse than cancer – it kills you in three to five years once diagnosed. You basically die of asphyxiation.


In the last week I stumbled upon another interesting paper on the effects of vitamin C on humans. A 2011 paper by Juraschek, et al. at Johns Hopkins University Medical School reported the results of a significant meta-analysis (a systematic review of multiple clinical trials) of 13 randomized clinical trials involving 556 patients who took a median dose of 500 mg of vitamin C per day. (I take a full gram)

The purpose of the study was to examine the effects of vitamin C supplementation on uric acid levels in the blood. Elevated uric acid levels in the blood causes gout, because saturation of blood with uric acid causes urate crystals to form in the synovial fluids of joints (e.g. crystal arthritis). Drugs that lower uric acid in the blood are used to treat gout because lowering uric acid causes the urate crystals to dissolve to ameliorate the arthritic pain.

Admittedly gout is not as bad as cancer. But another systematic clinical review of multiple trials on humans published in 2012 by Lottmann, et al. at the IGES Institut GmBH in Germany has shown clearly that having gout is associated with both all-cause mortality and, in particular, cardiovascular mortality. So what could be worse than death by gout?

I think I will keep taking vitamin C.

Alejandro Zaffaroni, 1923-2014

Alejandro Zaffaroni. (Life Sciences Foundation image)

Alejandro Zaffaroni. (Life Sciences Foundation image)

In a 1997 interview with Jill Wolfson and Tejinder Singh, Alejandro Zaffaroni shared what it was like for him growing up in Montevideo, Uruguay, where he was born on February 27, 1923. He described himself as “kind of a sick child” with asthma severe enough to keep him isolated from his peers. As a boy he spent his time alone outdoors playing, exploring, and thinking about what he found. And when not playing outside, he attended a Jesuit school where he rebelled against the strict disciplinary regime. Zaffaroni described this as an opportunity to “think a lot about all kinds of strategies,” a skill that came in handy in his adult life.

As Zaffaroni transitioned into a public school at the age of twelve, his mother passed away. His father, a banker, began to spend more time with the boy, exposing him to the cultural life of Montevideo through the symphony, opera, and other outings. Encouraged by his father to develop his own interests, Zaffaroni found chemistry and, with the help of a friend who had a much better grasp of the content than the rest of the class, found a subject he could excel in. Unfortunately, just five years after his mother had done so, Zaffaroni’s father also passed away. Yet his example continued to inspire his son.

As a pre-med student at the University of Montevideo, Zaffaroni encountered biochemistry, a subject he pursued further as the first Uruguayan to enroll in a biochemistry Ph.D. program in the United States. In July 1945 he headed to Rochester University which Zaffaroni chose over other options, including Harvard, because of the promise of freedom to follow his own research path in endocrinology, with a focus on steroids. After finishing his doctorate in 1949, he continued his work with steroids with the support of a grant from the National Institutes of Health and published his first article, “Adrenal Cortical Hormones,” in Science with Robert B. Burton and E. Henry Keutmann.

Finished with his education and ready for the next step, Zaffaroni had his pick of offers from several universities and private labs. Harvard again appeared to be a possibility, one that briefly brought him to the attention of Linus Pauling. In 1953, George. B. Kistiakowsky of Harvard wrote to Pauling for advice on a list of candidates for a new biochemistry professorship that included “Alessandro Zaffaroni.” Pauling underlined two names, Frank H. Westheimer, which he annotated with “best,” and Zaffaroni, which he annotated with “never heard of him.” Zaffaroni chose another path as Westheimer ultimately got the position, delaying further contact between Zaffaroni and Pauling for another fifteen years.


Continuing to follow his father’s earlier directive that he seek out what interested him most, Zaffaroni also turned down a position at the newly established Sloan-Kettering Institute which, according to a 2012 article in Life Sciences Foundation Magazine, had “one of the world’s top steroid labs.” Expanding on an established relationship with George Rosenkranz at Syntex, Zaffaroni chose instead to head to Mexico City to work for the smaller company, where he felt he would have fewer restrictions on his own research. At this time, in 1953, Zaffaroni also received his first patent, which concerned the extraction of adrenal hormones from bovine and porcine adrenal glands.

At Syntex, Zaffaroni worked on synthesizing steroids using a phytoestrogen extracted from yams. When he noticed that the quality of the yams interfered with the process, he went to the supplying yam farm himself and reorganized harvesting and transportation while also increasing its worker’s wages. Zaffaroni’s efforts were noticed by Charles Allen, who bought Syntex in 1956 and gave the young researcher a promotion. Zaffaroni and Rosenkranz quickly built the company into a major supplier of topical corticosteroids.

In 1962 Zaffaroni was named president of Syntex’s subsidiary in Palo Alto, California, and given the specific charge to gain access to the US pharmaceutical market. The company entered the nascent birth control pill market in 1964 and started creating offshoot companies to take advantage of Syntex’s various lines of research. This led to the creation, in 1966, of the Syva Corporation, which produced diagnostic equipment, as well as the pest control company, Zoecon, incorporated in 1968.

In addition to his executive duties, Zaffaroni also began thinking about new methods of drug delivery, but the culture at Syntex was not supportive of this work. So, funded by $3 million of his own money, Zaffaroni started ALZA in 1968 to focus on this new path of research. ALZA’s main products included an ocular insert designed to administer glaucoma medication, an intrauterine device for birth control medication, and transdermal patches, each of which incorporated timed release mechanisms. Though ALZA’s products were innovative, the pharmaceutical market was hard to move as eye drops, pills, and injections maintained their dominance.


The creation of ALZA, Inc., based in Palo Alto, coincided with Linus Pauling’s move to Stanford University in 1969. In March of that year, Zaffaroni introduced himself to Pauling, writing that he was “extremely pleased” that Pauling would soon be nearby and noting his eagerness to talk with Pauling in person about the burgeoning field of orthomolecular psychiatry. By enclosing some company literature, Zaffaroni also got Pauling interested in ALZA. The two met in April as Linus and Ava Helen were in the midst of their house hunting. The meeting was especially fruitful for Pauling as Zaffaroni provided him with a $100,000 grant to be divided over his next four years at Stanford.

For the next few years the two maintained an informal relationship by visiting each other, sharing ideas, and extending invitations to social gatherings. In 1974 Pauling brought a formal element to their relationship by asking Zaffaroni to become a member of the Board of Associates of the Linus Pauling Institute of Science and Medicine, at that point still in its infancy. Zaffaroni told Pauling in November, “I have been approached by many groups to participate in directorships of various worthwhile organizations, and have been forced to decline. But because of my great admiration for you and for your accomplishments, I am prepared to accept.” He warned Pauling that his busy schedule might interfere at times and in reply Pauling promised that he would not make too many demands. Zaffaroni’s fundraising experience quickly became central to his activities as an associate and he began a correspondence with Art Robinson – at that time LPISM’s Assistant Director – concerning the development of a prospectus for potential donors.

In the summer of 1975, Pauling asked Zaffaroni to extend his relationship with the Institute by joining its Board of Trustees. Again, Pauling promised that Zaffaroni’s duties would be minimal. Zaffaroni made one stipulation in his acceptance: that the Institute revisit the operation of its fledgling medical clinic. Zaffaroni told Robinson that the psychiatric research going on at the clinic needed a better review system. Robinson responded by suspending all outpatient services at the clinic. By November, with the changes in place, Zaffaroni agreed to join the Board.

In his new capacity, Zaffaroni continued his involvement in helping Robinson with fundraising. Their first outreach effort involved “two popular appeals,” one in Prevention and the other in Executive Health. The Institute published an article in both publications and placed a request for donations at the end of each one. Robinson reported to Zaffaroni in March 1976 that the Prevention article had generated 183 donations worth $5,101.50 while the Executive Health article received 103 donations worth $8,031.50.


(Life Sciences Foundation image)

(Life Sciences Foundation image)

Besides running ALZA and serving on the Board at LPISM, Zaffaroni continued his own scientific work. This included “Special requirements for hormone releasing intrauterine devices,” published in Acta Endocrinologica in 1974, and “Contraception by intrauterine release of steroids,” which appeared in the Journal of Steroid Biochemistry in 1975. He also received patents for his transdermal bandages in 1974 and 1976, and patents for controlled release tablets in 1976 and 1977. The following year he published an article, “Therapeutic Systems: The Key to Rational Drug Therapy,” in Drug Metabolism Reviews, that described some of his new developments.

Pauling tried to tie in his own interests to these drug delivery systems. In March 1979, Pauling wrote to Zaffaroni about a study on the improvement of patients with anorectal cancer who were given time released capsules of ascorbic acid. Pauling saw the improvement as being due to topical effects and shared his idea for a similar “slow release” capsule for stomach cancer, which was then afflicting Ava Helen. Pauling’s capsule would “spring into the shape of a sphere, which would cause it to be retained in the stomach” as the ascorbic acid was released into the stomach and afterwards broken down and digested. Zaffaroni responded that ALZA was already at work on something similar to what Pauling described and that he was eager to talk more about it. By 1986 ALZA had released a “once-a-day” Vitamin C supplement utilizing its controlled-release technology.

Amidst the biotech boom of the early 1980s, Zaffaroni’s business practices came under closer examination. In 1982 Time highlighted Zaffaroni’s recent start-up, DNAX, as one of the genetic engineering companies “having trouble living up to their early billing” as one of the “hottest companies on Wall Street.” The article pointed out that to start the company Zaffaroni had “easily raised $5.5 million,” but was now “spending nearly $4 million annually on research, and…does not expect to see any profits for at least another six or seven years.” Other sources in the popular press were often more critical, focusing on Zaffaroni’s commercial, rather than scientific, performance.

In August 1985, Zaffaroni decided to resign from LPISM’s board. He felt that his work was done, telling Pauling that the Institute “has now attained worldwide recognition” and that, with Pauling’s “guidance it will continue to evolve in many positive directions.” He continued

My pattern, as you know, has been to participate in the founding of various enterprises and, once they are established, to go on to new ones. That is because I believe that what is done initially counts more than anything else. Thus, my focus has generally been on contributing innovative concepts at the outset rather than remaining permanently associated with any particular endeavor. That pattern enables me to do what I do best and to keep from becoming stale. It also leaves room for others to follow with fresh insights and new concepts. Thus, I believe it serves everyone well.

I am sure that you know, without my saying it, that the main inducement to my acting as Trustee has been the opportunity it gave me to work with you. That association has brought me great personal pleasure, intellectual challenges, and a keen appreciation of your many gifts of heart and head. You may be sure that I stand ready to help you at any time in any way I can, should the need arise.

Zaffaroni did, however, remain on the Institute’s Board of Associates until 1996 and maintained his connections to LPI into the 2000s when he met with longtime administrative officer Stephen Lawson in Palo Alto to discuss the Institute’s collaborative research on ALS and peroxynitrite in Uruguay.


Congratulatory note from Pauling to Zaffaroni on the occasion of Zaffaroni being honored by the Weizmann Institute of Science, November 1989.

Congratulatory note from Pauling to Zaffaroni on the occasion of Zaffaroni being honored by the Weizmann Institute of Science, November 1989.

After leaving LPISM, Zaffaroni also stepped down as ALZA’s CEO to seek out new endeavors. In 1988 he asked Pauling to become the Honorary Scientific Advisor for the newly forming Affymax Research Institute. As part of the deal, LPISM received 25,000 shares of stock in the company. By June 1990, Zaffaroni told Pauling that Affymax was “moving from an early stage ‘start-up’ to a successful development stage pharmaceutical company.”

The press continued to be somewhat cautious. The following year, the New York Times still referred to Affymax as a promising start-up, but was concerned with Zaffaroni’s avoidance of investment bankers by relying on his many contacts to raise his own funds. The Times followed up with questions as to how Affymax “burned through over $20 million so fast” and by noting that its central product -VLSIPS, short for Very Large Scale Immobilized Polymer Synthesis, which were biological compounds produced on silica chips developed by the semiconductor industry – was not selling well. Zaffaroni was not concerned with Affymax’s profitability at all, informing Pauling in May 1991 that he had raised $26 million and was ready for the company to “aggressively pursue our scientific and commercial goals” as well as move into their new research facility. By the end of the year, Affymax began offering public stock.

At the end of the 1990s, Zaffaroni began attracting more praise in the press, particularly for his founding, in 1995, of Smyx, a company focusing on the applications of combinatorial chemistry in the development of drugs. But he was also running up against new criticism echoing that issued in the early 1980s about the viability of DNAX. This time those fears were directed at Affymetrix, which had been spun off from Affymax in 1992 to focus on VLSIPS. An article in Forbes reported that the chips, though potentially “a godsend to medicine,”  may not be a “godsend…to Affymetrix’s bottom line.”

Zaffaroni soon had more direct problems to deal with. In 1995 he organized the sale of Affymax to Glaxo for $533 million. Some of Zaffaroni’s friends and family members began trading shares of Affymax just before the sale was announced publicly, which drew the attention of the Securities and Exchange Commission. Charged by the SEC with insider trading, Zaffaroni and six others agreed to pay fines of $1.85 million and immediately tried to move on. According to the New York Times, Zaffaroni’s lawyer stated that his client wanted “to devote his time to science and charity rather than litigation.” And that’s just what he did as he continued to produce more patents related to drug delivery and VLSIPS.

The 2000s saw Zaffaroni open up another line of research, this time for a drug delivery system that used the cigarette as a model. In 2000 Zaffaroni started Alexza to focus on this research and, before too long, he, along with Joshua D. Rabinowitz and Dennis W. Solas, produced patents for delivering insomnia, anti-inflammatory, antipsychotic, pain relieving, and several other drugs through inhalation. Unlike other inhalation drugs, these relied on the slight heating of the drugs before delivery. Zaffaroni and others described their research in “Fast Onset Medications through Thermally Generated Aerosols” for the Journal of Pharmacology and Experimental Therapeutics in 2004. In 2012 Alexza’s first product, aimed at schizophrenia and bipolar disorder, was ready for market. That same year, at the age of 89, Zaffaroni was inducted into the National Inventors Hall of Fame.


Zaffaroni passed away this past March 1st from complications related to dementia. When asked in 1997 how he wanted to be remembered, Zaffaroni said

Well, the one thing that was always very important in my life is human relations. So in looking at candidates for any of the jobs that I had, I wanted to have people who shared my value system, in addition to being the kinds of capable individuals that I needed to have.

Perhaps drawing on his father’s support and encouragement of him as a youth, Zaffaroni continued,

It is tremendously important in building this company that I create a very, very warm caring environment, so that people have an opportunity to do the best of their work. One of the key things to success is never to worry about failing. Many people do not do a lot of the things that could be done because they do not want to have a negative result. If you don’t go for the new breakthrough, if you are going just to stay in the areas which we all know, we are stationary.

Now on the other hand, if you make a huge effort with a new idea and you don’t succeed, the big companies don’t see that as a good thing. So why take risk, if there is no opportunity to be rewarded by the effort?

In my view, the only thing attractive in life is continually to move forward, to be looking for new opportunities, and to support people and let them fail safely.

Pioneering the Field of Proteomics

John Leavitt, 1982.

John Leavitt, 1982.

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

In the fall of 1985, I went to a small meeting in Heidelberg, Germany, with Steve Burbeck from the Linus Pauling Institute of Science and Medicine, who had helped me by developing computerized microdensitometry to analyze two-dimensional protein profiles. At this meeting I described our protein profiling work and the discovery of the mutant beta-actins and another interesting protein which I named “plastin.”

Steve Kent, head of the protein sequencing facility in Leroy Hood’s lab at Caltech, heard my talk. We sat across from each other at dinner and he proposed a collaboration to develop methods of sequencing minute amounts of protein leached from spots in high resolution protein profiles. Lee Hood was well known for developing state-of-the-art protein and nucleic acid sequencing methods and machines, and was a founder of Applied Biosystems in Foster City, CA.

After I returned from Heidelberg, Ruedi Aebersold called me from Caltech and we began collaborating on microsequencing of pure nanomolar quantities of unknown proteins of interest eluted out of my protein profiles. In this work we essentially started the field of proteomics, which was eventually named ten years later by Jim Garrells, a protein profiler at Cold Spring Harbor. Proteomics is the search for and definition of proteins that could serve as diagnostic markers and drug targets for diagnosis and treatment of diseases, in our case cancer.

In 1987 we published a landmark paper in PNAS on the microsequencing technique that Ruedi developed. This paper would eventually be cited in references by more than 1,000 other research papers.

Ruedi

I gave a postdoctoral fellow, Mahdu Varma, the task of isolating the cancer-specific leukocyte isoform of plastin (L-plastin) from 140 protein profiles. This protein has been implicated in metastases in both melanoma and prostate cancer as well as in other aspects of cancer. The L-plastin spot was easily recognized and those spots on a nitrocellulose filter were “snipped out,” removing all the other proteins of the cell. We sent Ruedi a plastic tube containing the 140 “spots” of L-plastin. He had figured out a way to solubilize the protein from the nitrocellulose and was successful in determining the sequence of eight oligopeptides of between eight and sixteen amino acids derived by digestion of L-plastin with a proteolytic enzyme.

The peptide sequences he determined turned out to be perfectly accurate internal amino acid sequences of plastin when we decoded the sequence of the plastin gene (cDNA) clone, a reverse transcript of the messenger RNA. This was the first time that anyone had done this and it opened up the field of proteomics and led to the discovery of other diagnostic and drug targets.

plastin 1

We had chosen L-plastin, normally only expressed in white blood cells, because I had reported for years that it was a cancer marker in tumors that arose in solid tissues (identified in the image above by the two upward arrows). After we received the oligopeptide sequences from Ruedi, we made short DNA antisense probes that would hybridize to DNA sequences encoding these peptides in the human genome to fish out the full-length DNA clones that carried the sequence of the L- plastin gene.

Ching Lin and I, along with Reudi, published the sequences of the human L- and newly discovered T-plastin proteins, based upon sequencing of cDNAs, in Molecular and Cellular Biology. The discovery of a second isoform of plastin (T-plastin named for tissue plastin as opposed to L-plastin from leukocytes) was a surprise. We now had two genes to characterize at the genomic level. Today, T-plastin is a well recognized marker for cutaneous T-cell lymphoma (Sezary Lymphoma) and L-plastin, inappropriately expressed in solid tumor cells (carcinomas, fibrosarcomas, etc.), is understood to be a contributor to metastasis.

The Linus Pauling Institute was not all work and no play in the 1980s

We worked hard at the Institute and Linus Pauling was always there and visible.

We put together a softball team with Jim Fleming, Dan McQueeny, Zelek Herman, myself, and others at the Institute and played departmental teams at Stanford. I think we were called the “Pauling squeeze.” After these games we would often go dancing at the Class Reunion on El Camino Real near the corner of Page Mill Road.

We were fortunate to have on staff a first rate fundraiser in Richard (Rick) Hicks who arranged wonderful parties on Nob Hill at the Stanford Court. The most memorable of these parties occurred in late November 1986, when we honored Japanese billionaire Ryoichi Sasakawa with the annual Linus Pauling Medal. Another year Carl Sagan and Ann Druyan, who helped Carl put together the Cosmos series, likewise took part. We often saw Dr. Pauling’s sons, Linus Pauling Jr., Peter, and Crellin as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

In 1988 I moved on to a new position in San Jose and then became Director of Research at Adeza Biomedical. Since we continued to live in Palo Alto, we continued to interact and party with the Linus Pauling Institute staff into the 1990s.