My profession as a Molecular Biologist must seem like an odd pursuit to many. In late 1977 and 1978 my career as an independent researcher started to emerge at Johns Hopkins with my publication of two original research papers in Nature on phenotypic behaviors of cancer cells. These papers made it possible to nail a job at the National Institutes of Health as a Senior Fellow at the Bureau of Biologics with the FDA. It didn’t take me long to capitalize on this freedom and what seemed like unlimited funding to pursue the research that I dreamed up and wanted to do. The following tells what happened next.
John Leavitt, 1988, at the Pauling Institute
There was an article about Linus Pauling in Time magazine in early 1981 about the fact that at the age of 80 he was still seeking a grant from the National Institutes of Health to fund his research on ascorbic acid for treating diseases. This news caught my attention and I looked into the possibility of joining Dr. Pauling’s institute. Toward the end of the summer I was invited to visit the Pauling Institute in Palo Alto, CA to give a seminar on my research at NIH.
In late August, Koloman Laki, an aging scientist at NIH, called me up and invited me over to his lab in NIH Building 10, a short walk across the campus from my lab in NIH Building 37. He was interested in talking to me about my recent discovery of mutations in human non-muscle cytoskeletal actin that was published in Cell in late 1980. This protein is the major architectural protein of all eukaryotic cells and we had shown that it was the most highly conserved protein in evolution of the species from yeast to humans. This fact made these mutations even more interesting.
Koloman was a protege of the Hungarian Nobel Prize winner Albert Szent-Györgyi who, I later learned, was much admired by Dr. Pauling because he had discovered both vitamin C and actin. Koloman described how Szent-Györgyi discovered muscle actin. When I mentioned that I was to visit the Linus Pauling Institute in late September, he told me about Emile Zuckerkandl’s and Dr. Pauling’s work on the ‘biological clock,’ which provided evidence in support of Charles Darwin’s theory on divergence of the species.
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 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.
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 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 (mass) 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 most abundant of which were cytoplasmic beta- and gamma-actin, at a ratio of about 2:1 in abundance, respectively. The figure 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 appeared at about one unit 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.
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 Gottingen, 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 Vandekerkhove, 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.
Flash forward to the fall of 1981. In the last week of September, I flew to Oakland, CA and was picked up at the airport by Emile who was President of the Linus Pauling Institute of Science and Medicine. The next morning I stood up in front of Dr. Pauling and the institute staff to tell them about my discovery of a mutant human beta-actin and my speculation on its involvement in neoplastic transformation. The evidence suggested that I had actually discovered at least two mutations in the same gene, each of which caused a progression to a higher malignant state. Dr. Pauling could relate to the way I made this discovery because he was the first to describe a mutation in a gene that caused a disease, in his case a mutation in charge-altered hemoglobin that caused Sickle Cell Anemia.
Linus Pauling was in the front row and was all smiles. He asked me if I knew who discovered actin. I was prepared to answer that question thanks to Koloman Laki. In the afternoon I met with Emile who offered me a Senior Scientist position at the Institute, which I accepted. At the time it would be me and Dr. Pauling with separate research interests.
So I resigned my secure job-for-life at NIH (I had been promoted to a civil service position from my Fellow position) and moved to Palo Alto to join the struggling Linus Pauling Institute. My technician, Patti Porecca, hired from Bob Gallo’s lab at NIH, would follow me to the Pauling Institute.
Cloning of the Human Beta-Actin Gene
After I arrived at the Pauling Institute, two of my colleagues at NIH and I published a comprehensive study of the changes in protein expression between normal and neoplastic cells in Carcinogenesis using high-resolution computerized microdensitometry to analyze the complex protein patterns (my first paper from the Pauling Institute). This was the first time that such a study had been published, e.g. the comparative profiling of expression of a large number of proteins in neoplastic cells. It was a study of the 1,000 most abundant proteins in normal and neoplastic human cells which revealed potential biomarkers and causative genetic events for human cancer. At the time it was staggering to view these patterns but perfect for my dyslexic brain and mind’s eye. In addition, we had published another paper in Cell that described, for the first time, the progression of a neoplastic human cell to a higher malignant cell following a second mutation in the same beta-actin gene. Early in 1982, Steve Burbeck and Jerry Latter at the Institute set up the same computerized microdensitometry platform I had exploited at NIH.
Jerry Latter gave a stirring talk at Argonne Labs in Chicago demonstrating that computerized microdensitometry of protein profiles could be used to determine the identities of unknown proteins based upon determining their amino acid compositions in situ in protein profiles. This paper was published in Clinical Chemistry in 1984. At the same meeting, Steve Burbeck described a truly innovative invention that could measure beta-particles emitted from radioactive protein profiles to produce a direct image of the protein profile pattern. As a group we had entered an exciting period of discovery and innovation at the Linus Pauling Institute.
Shortly after I arrived in Palo Alto in December 1981, I called Professor Larry Kedes at Stanford and we embarked on a collaboration to clone the human beta-actin gene. His impressive postdoctoral fellow, Peter Gunning, taught me some basic recombinant DNA techniques, and I was off to the races. The difficulty was to identify the functional gene in a sea of actin pseudogenes (sometimes referred to as junk DNA). I used an elegant method of homologous recombination developed in Tom Maniatis’ lab at Harvard that had never been used before to clone a novel gene (In fact, cloning of human genes was just getting started at the time). This was smart because Professor Maniatis would be the chairman of the NIH study section that reviewed my first grant proposal submitted from the Pauling Institute. I did not know it at the time but within a few months after starting this work, I had cloned the functional beta-actin gene a week before Christmas in 1982.
I developed a scheme to identify the correct gene among 300-400 clones of pseudogenes that Patti and I had cloned and the strategy worked. We gave Dr. Sun-Yu Ng the task of sequencing the DNA clone that we were betting on. Rather quickly we determined that we had cloned the functional human beta-actin gene because the DNA sequence that Sun-Yu determined from our candidate clone accurately encoded the amino acid sequence of human beta-actin protein that I had published in Cell in 1980 with Klaus Weber. Quite coincidentally another lab discovered a rat oncogene that was a fusion of part of an actin gene with a tyrosine kinase gene. I sent this information off to the study section that was reviewing my grant in January 1984 as added evidence that the actin gene was in some way relevant to neoplasia.
My colleagues and I at the Pauling Institute and Stanford published our successful isolation of both the mutant and wildtype human beta-actin genes in in October 1984. As shown on the left, we had given Armand Hammer’s name to our cancer research program because of his generosity in helping to fund the Linus Pauling Institute.
In January 1984, I was awarded a grant of about $110,000 a year for two years from the American Cancer Society…what a relief. Later in the spring I received word from Professor Maniatis’ NIH study section that our program would also be funded in June by a grant of about $150,000 a year for 3.5 years from the National Cancer Institute for the same work (this grant was successfully renewed twice for six more years through 1994). I was able to hire Dr. Ching Lin from Iowa State University and Dr. Ng (Sun-Yu) from Kedes’ lab. By 1985 Sun-Yu finished the complete DNA sequencing of the human beta-acid gene and Ching sequenced the copy of the beta-actin gene that had two mutations to formally prove the mutations at the level of the gene. Everything that we had learned about the genetic code and amino acid sequences of proteins made our findings predictable. I had learned from my own research how Darwin’s theory of evolution and natural selection worked.
This was the year I finally successfully transferred in recombinant gene inside a cell in culture. I transferred the mutant human actin gene into a rat fibroblast cell line to show that I had cloned the functional gene which could abundantly express its protein the way the natural endogenous beta-actin gene worked (shown in a protein profile of these actins above).
At this point I had a brief meeting arranged by Emile with Alex Zafferoni, founder and CEO of Alza Corporation (among other successful companies), a block away on Page Mill Road. Zafferoni recommended Bert Roland as a patent attorney. I arranged a meeting with Roland, also a block away, for that afternoon to discuss patenting the human beta-actin gene promoter because of its strong constitutive nature (the engine of the gene that drives its expression and regulation). I told Bert that this was a collaboration with Peter Gunning and Larry Kedes at Stanford. Roland was famous for filing Boyer’s and Cohen’s genetic engineering patent which created Genentech and eventually funded Stanford with hundreds of millions of dollars in royalties.
We published Sun-Yu’s work on the sequence, structure, and chromosomal location (chromosome 7) of the human beta-actin gene in Molecular and Cellular Biology and we published Ching’s work locating three mutations in this gene in the Proceedings of the National Academy of Sciences, sponsored by Linus Pauling. A patent was filed on the beta-actin promoter and over the years it was licensed to about 15 biotech companies by Stanford University. This patent was prosecuted and licensed for the full 17 years (the life of a patent then) but the patent never issued. The Institute’s first royalty check was about $10,000 in 1986, but most of the royalties were earned by Stanford’s patent attorneys.
Peter, Larry and I published a paper in PNAS on the use of the human beta-actin gene promoter for expression of other genes. This vector was distributed to anyone who asked for it – and many did – and to those companies that licensed the invention. At last count this paper had more than 1,000 reference citations.
Our paper popularized the actin promoter as a strong constitutive promoter of foreign gene expression. Soon the rice actin promoter would be used to make Round-up Ready crops by DeKalb Genetics and Monsanto, and giant tilapia fish would be engineered with growth hormone under the control of the fish beta-actin promoter. There were even fluorescent mice running around in Japan created with firefly luciferase expressed by the beta-actin promoter (which I called “the cat’s meow”). Since cytoplasmic actins are the most abundant proteins in most cells you could use the promoter to abundantly express foreign genes in most cells of any animal.
In 1987 we also published the culmination of my research on the mutant beta-actin gene in Molecular and Cellular Biology. When I introduced this gene into non-tumor forming immortalized human fibroblasts they became tumorigenic. The results showed that the more abundant the expression of the mutant beta-actin, the more tumorigenic the non-tumorigenic cells became and the cells that came out of the tumors were enhanced further in the level of mutant beta-actin expression. This was a sensational finding that was the goal of research which began with the discovery of the mutant beta-actin in 1978 at NIH.
The Mutant Beta-Actin Effect was Reproduced and Extended in 2013
John Leavitt in his laboratory at the Linus Pauling Institute of Science and Medicine. Originally published in Science Digest, June 1986.
As stated above, 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 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 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. 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 of this mutation’s 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:
- 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);
- While the mutant actin concentrated primarily in these ruffles at the edge of the cell green palloidin staining revealed the location of normal filamentous actin in stress fibers (right image);
- The expression of mutant actin inhibited the tropomysin binding to filamentous actin and tropomysin did not accumulate in the ruffles; and
- Mutant actin co-localized with Rac1 (a GTPase mediator of membrane ruffling) and beta1-integrin (adhesion protein) in the ruffles.
The other surprising finding in my own lab was that cell lines expressing the transfected mutant beta-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 at the same level 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 after we reported it.
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 in my lab at the Pauling Institute), 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.
Pioneering the Field of Proteomics – Discovery of Human Plastins
In the fall of 1985, I went to a small meeting in Heidelberg, Germany, with Steve Burbeck from the Pauling Institute, 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 among all of the proteins made by cells 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.
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 to the left. So I named the protein “plastin” – the greatest new thing since sliced bread.
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 now been implicated in metastases in both melanoma and prostate cancer and more recently in breast and colon carcinoma cells. The L-plastin spot was easily recognized and those spots in gels were transferred to nitrocellulose filters, stained, and “snipped out,” effectively 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 internal oligopeptides of between eight and sixteen amino acids derived by digestion of L-plastin with a proteolytic enzyme.
The peptide amino acid sequences Ruedi determined turned out to be perfectly accurate internal amino acid sequences of plastin when we decoded the sequence of the plastin gene (cDNA) clone from a reverse transcript of the messenger RNA. This was the first time that anyone had done this; this opened up the field of proteomics and has led to the discovery of other diagnostic and drug targets by the same method. A lot of this work was done by Reudi Aebersold because he was flooded by requests for help.
We had chosen L-plastin, normally only expressed in white blood cells (L for lymphocytes), because I had found over the years that this polypeptide was expressed in tumor cells that arose in solid tissues (identified in this electrophoretic image on the left by the two upward arrows), but this protein was absent in the normal cells of tissues in which the tumors arose. After we received the short peptide sequences from Ruedi, we made short DNA antisense probes that would hybridize to DNA sequences encoding these peptides in the human genome in order to fish out the full-length cDNA clones that carried the sequence of the L-plastin gene. Dr. Ching Lin 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 radioactive probe, then we would perform a quick test to determine that we had cloned the L-plastin coding sequence. But science is full of surprises and we found that the first clone Ching 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 new clonal DNA as a new radioactive probe against the tumorigenic fibroblast cDNA library. He isolated another clone that passed the test and detected a gene that was expressed in lymphocytes and tumorigenic fibroblasts but not in normal human fibroblasts.
Ching (to the left), Madhu, and I, along with Reudi, published the nucleic acid sequences of both clones, the human L- and newly discovered T-plastin proteins, by sequencing of their cDNAs, in Molecular and Cellular Biology in 1988. 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-established marker for cutaneous T-cell lymphoma (Sezary Lymphoma, a lethal white blood cell cancer) and L-plastin,which is inappropriately expressed in solid tumor cells (carcinomas, fibrosarcomas, melanomas, etc.). L-plastin and T-plastin are now implicated in many forms of tumor metastasis. It is metastasis that kills us when we develop cancer.
We continued to work on the two plastins and published the full genomic sequences and their chromosomal locations in 1993.
Extension of Our Research on Plastins by Other Labs
The figure below maps the progression of discovery that followed our research on plastins, which began at the Pauling Institute in 1985. The number of our publications by year is shown in red in the graph and research papers published by other labs is shown in the blue bars up through June 2014.
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 papers were published between the fall of 1985 when we coined the name plastins and June 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.
The Linus Pauling Institute was not all work and no play in the 1980s
We worked and played hard at the Institute and Linus Pauling was always there and visible.
We put together a softball team with Jim Fleming, Dan McQueenie, 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 fundraising 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, took part. We often saw Dr. Pauling’s sons, Linus Pauling Jr., Peter, and Crellin at these get-togethers and at the Pauling Institute as well.
Here we are at the Stanford Court that night in November 1986 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 become Scientific Director of the California Institute for Medical Research in San Jose and then became Director of Research at Adeza Biomedical in 1991. I spent my final year in 1995 as a Molecular Biologist on contract from the Palo Alto Medical Foundation to the US Air Force Academy in Colorado Springs, where I looked into the mutagenicity of high energy short pulse laser light that was used in the battlefield. I was ready to move on to a new line of work consulting with Biotechnology and Pharmaceutical companies, which lasted for 19 additional years.