Friday, December 27, 2019

Graduate School in Wild Texas--Yeeha!

Texas Beach Scene--somehow I earned a PhD in three years

         In early January 1974, I flew down to Texas from my family home in New Jersey. My one suitcase was filled with woolen winter clothes, leather boots, and only a few summer things. I brought my pillow and a typewriter.  Professor Pat Parker picked me up at the airport in his old VW bug, rusted out on the sides. He was a modest looking man with a small mustache, a bit of a limp, and shaggy brown hair. Even as the Director of the Institute, he wore old khakis and an un-ironed short sleeve shirt with no tie. I had formed a different picture of how he looked based on his Science papers that I had read at Penn State.

         We drove to Port Aransas on Mustang Island, where the Marine Science Institute was located, with the final portion of the journey via ferry. Coming from urbanized Southern New Jersey, Port Aransas seemed like a town literally at the end of the earth for me. The next day I met Professor Chase Van Baalen. Chase was often mistaken for one of the maintenance staff. A short fellow with a crew cut, Van Baalen smoked a pipe, and was known for shoving his lit pipe deep into the pockets of his jeans. His right hand lab assistant, Rita O’Donnell, took care of the lab glassware and culturing facility for Chase. She was a crusty Port Aransan, who also owned and managed a popular motel with her husband. Parker, Van Baalen, and Rita formed the core of my scientific mentors at the Institute.

            My first house in Port Aransas was a two-room bungalow within walking distance of the Marine Lab. I bought an old bicycle that I used to get to town for shopping. It was the first time I had lived alone. I needed to buy plates, pots and pans, silverware in one of the two grocery stores on the Island. Without a car, I couldn’t get to the larger supermarkets and stores on the mainland. Meals were generally one-pots. My specialty was rice-a-roni with vegetables and an egg scrambled in at the end. Life was pretty simple. 
My first bungalow in Texas, this photo taken in 2011

         I settled in quickly, meeting and getting to know other graduate students and postdocs. My first mini-project was to assay naturally occurring iron binding molecules made by microorganisms. The availability of iron limits the growth of almost all organisms from microbes to humans. Humans often take iron supplements in their vitamin pills. In the natural environment, organisms have had to figure out how to do this on their own.  We were searching for new compounds that assisted bacteria and algae to easily take up iron without being limited. Others had found these molecules in cultures, but no one had looked for them in nature.  I gathered algal mats from nearby lagoons and shallow marine sediments, extracted them, and then placed the extracts on a lawn of bacteria that required an iron-binding compound to grow. We discovered that organic, iron-binding compounds were widespread from microbes in the natural environment, not just secretions from laboratory microbes. It was a winning small project, and the subsequent publication, standing the test of time, is still relevant (Estep et al., 1975).   
UT's Marine Science Institute

            In 1974 when I started grad school, the Vietnam War was winding down. Mustang Island, Port Aransas, Texas, where the Marine Science Institute was located saw an influx of returning soldiers who had discharged out of the service and gravitated to South Texas for a number of reasons, one of which was easy access to a pipeline of heroin coming in from Mexico. Jobs were easy to find on shrimp boats, in construction, or on oilrigs in the Gulf. Being an academic in this small town meant you were in the minority and a world away from the city life of Austin, the main Univ. of Texas campus.

            I met many of these vets in the local “Port A” bars, the only social places on the island where people hung out after work. Compared to State College, Pennsylvania or New Jersey the folks that lived on Mustang Island were a rougher crowd than I was used to. Shorty’s bar down on the Flats of Port Aransas near the docks was a rough and tumble place run by a 70 year old woman, Shorty, who kept a pool stick and a pistol behind the bar to keep order. A gruff bartender, she knew all of her customers and lent them a $10 bill when they needed it or gave a free beer to someone who couldn’t afford the $1 that she charged for a draft. Sadly, a drunk customer who didn’t understand her brand of “humor” murdered Shorty in her bar in the 1990s.

            Next door to Shorty’s was The Party House, owned and operated by a former Marine Science grad student and some buddies. It was a raucous place filled to the brim with the younger hippy crowd. They served pizzas—the only place in town where you could get one. It was common for the rowdy customers to snag pieces off of your pizza as the waiter brought it to your table. By midnight, everyone howled with the music, yelled “Yeeha!” and danced wildly. I learned how to dance the Texas classic dance--the Cotton Eyed Joe--there, which generally erupted in the wee hours.

            Port Aransas in those days boasted one of the widest, cleanest beaches on the Gulf Coast. The sand was firm enough to drive on, so the beach was a scene of Texans cruising the surf line in their fancy pickup trucks and Cadillacs—a far cry from the Jersey Shore where I had grown up. People brought their BBQ grills with them along with coolers of Lone Star, Pearl, and Shiner beer. By nightfall, fireworks lit up the sky and the smell of marijuana wafted from most of the vehicles. Camping was permitted on the beach either in RVs, tents, or makeshift encampments around trucks. Although sheriffs cruised the beach day and night, it was decidedly a “scene”, which contrasted to the quiet, rarified atmosphere at the Institute.         

            Soon after I arrived I met Carlos Jack Estep in the Sail Club saloon, a hangout for the country-western crowd. Jack used the old line, “What’s a nice girl like you doing in a place like this?” Country music blared on the jukebox and we danced to “Behind Closed Doors,” a popular ballad by Charlie Rich. He was thirteen years older than me, had been married before, and was known all over Port Aransas as Jack the Sign Painter. With a shock of straight blonde hair and a sly smile, he had many of the local Port Aransas lasses following him around. He’d served in the US Air Force, had had a life as a businessman in San Antonio, but now was more of a hippie who traveled to Central America and took it easy. At that time, I had no aspirations of becoming a middle class person like my parents. The idea of an alternative life style was attractive.

            Alone and not yet in step with my lab colleagues, I was swept into a whirlwind romance. One month later, we eloped and got married on a party boat that took tourists out to fish in the Gulf for the day. At the time, I thought this was very romantic and adventurous. I was out on my own and making decisions. Years later, I could see that a wedding without family is a red flag for future trouble. At that time, young people were choosing to live in different ways than their parents, typically without marriage. I thought my new moniker as a “wife” was cool, but also risky and a bit reckless. I’d never done anything reckless in my life. I took a chance here. It was a very heady time.

            I kept the hasty marriage a secret from my family and friends until at least a month later, guessing that the others might not be as thrilled about it as I was. I ended up writing to my parents telling them about the marriage. My family was ripped apart by the news. My mother and my sister Barb flew down to Texas for Barb’s spring break and met Jack. He was on his best charming behavior, showing them some of the local Texas sites, barbequing great meals, and telling jokes. The shock of a secret wedding was smoothed over.

            As a newly married woman, I could concentrate fully on my work in the lab rather than pay any attention to the interlab gossip about who was dating whom. I arrived at work by 8 am, kept my head down until 5:30 pm, and then pedaled my bicycle back to the little house we lived in for the first half-year. Grad students were required to move to Austin for two semesters to take formal classes, so we were uprooted from the beach life to big city Austin in August 1974. Rather than rent an apartment, we had the “brilliant” idea to take an old cab-over camper and convert it into a houseboat that would reside at a marina on Lake Travis, in the hill country west of Austin. Jack started the remodel, but with just about everything he started, his work rarely lived up to expectations and frequently never was completed. Our “house boat” became a small land trailer that lacked a bathroom. The only running water was a hose connecting to a small sink in a tiny kitchen. We lived in this modified camper for not quite three years, occupying various trailer parks in Port Aransas after returning from Austin.

            On main campus in Austin, I spent fall semester as a teaching assistant for Oceanography, earning about $300/month. Jack was a sign painter by profession, with no connections in Austin, so my salary provided our total support. We qualified for food stamps, which kept us going. I also had a side business making jewelry out of used, old eye glass lenses, something I had been doing since I was a college student. Between my studies, I made pendants and pins adorned with laminated famous paintings that Jack sold on the streets at craft markets. A necklace sold for $4 and a pin for $3. We made just enough to get by. In winter 1975, I needed to spend time studying for my qualifying exam, a nerve-wracking oral examination designed to find out everything that you didn’t know. I also completed my course work and passed the German foreign language exam by reading a hefty tome by Otto Warburg, a German physiologist. No question we were living in poverty, but that mattered little because I was consumed by my work.

            Returning to Port Aransas at the end of May, there was nowhere to park the “houseboat” so we lived on the beach for a couple of weeks before finding out about a small tourist motel with eight units that needed to hire managers. We applied and became managers of the Gulf Beach Resort Hotel situated half a block from Port Aransas’ white sand beaches. We were then able to move into a small apartment with air conditioning for the summer. 
The Gulf Beach resort motel (in 2012--now pink)

            Before we’d moved back to Port Aransas, we purchased a 1966 International Scout that had been used as an ice cream truck in Austin. Down in Port A, Jack painted the truck with pink and white stripes with the Pink Panther cartoon character eating a popsicle on each side.  There was already one ice cream truck selling on the beach so we named our truck “The Other Ice Cream Truck.” We purchased a second large floor freezer and once a week the ice cream supplier from Corpus Christi loaded it up with various ice cream products. We hired two young folks to drive the truck---a buxom blonde who wore a bikini and a good-looking blonde surfer dude with a charming easy personality. It was not long before I became the number one ice cream dealer in South Texas!

         I started my PhD project in summer of 1975 with funding from a NASA Exobiology grant working with Parker, Van Baalen, and microbiologist/enzymologist F. Robert Tabita. My PhD research was to purify the enzyme key in fixing carbon dioxide in photosynthesis—Rubisco--from various organisms, then carry out a series of complicated experiments to measure how carbon isotope patterns were modified by Rubisco.

         Harmon Craig—one of the earliest pioneers (an Isopope to be sure) in stable isotope research--wrote the first blockbuster paper on “stable carbon isotopes in everything” from diamonds to an unwitting discovery of a different type of grass with a unique carbon isotope signature (Craig, 1953). His later work tracked major carbon isotope systematics in the world’s oceans. At the same time, Melvin Calvin and A. A. Benson were using radiocarbon to track the pathways of carbon fixation (Calvin and Benson, 1948) that would eventually garner Calvin a Nobel Prize. A paper by Park and Epstein (1960) linked the two types of studies and measured, for the first time, the carbon isotope fractionation by the CO2 fixing enzyme Rubisco. Using a purified enzyme, bicarbonate, and ribulose 1,5-bisphosphate (RuBP), they calculated an isotope fractionation indicating the lighter isotope of carbon was favored over the heavier one during the reaction. Not much was known about the structure and function of Rubisco at this time.

         Work in the late 1960s and early 1970s on the structure and function of Rubisco brought new interest to the carbon isotope fractionation studies in plants. Bob Tabita, then a recent PhD and postdoc, brought his skills in purifying Rubisco as an Assistant Professor at the University of Texas. His work centered on isolating and describing the different forms of bacterial Rubisco enzymes—their structures, kinetics, and activations. Rubisco from higher plants has a very large molecular weight (550,00 daltons) and is composed of 8 large subunits and 8 small subunits.

            In June at the Laboratory, I took my oral exam and was asked questions about lipid (fat) metabolism, a topic I did not study thoroughly. After writing an essay on that topic, the exam was passed and I became a PhD candidate. I had to tell my professor Chase Van Baalen that I had another job, or two. His response was, “As long as this doesn’t affect your work and you’re here Monday through Friday, it’s fine with me.” I kept my nose to the grindstone. Van Baalen did not believe in chairs in the laboratory, so I stood all day at my assigned lab bench. He also did not believe in automated biochemistry equipment, so all of my enzyme purifications were carried out by manually advancing a rack of test tubes every 5 minutes. 

            Van Baalen had several folksy expressions he liked to use. If you were found sitting at your desk, instead of at your lab bench, he’d pop his head in to your office and ask, “Wanta learn a trade?” He wore a slightly devilish little smile. The answer you needed to give was, “Sure.” One time when I was asked this, I was taught how to change the oil in a vacuum pump. Changing a pump’s oil is a messy, slimy job that nearly everyone avoids until it absolutely has to be done. I learned two things from him—a technical skill I have used for my entire career—and a management tool to get timid students to try something new. After a couple of months of figuring things out how things worked in the Van Baalen laboratory, by the fall semester I was well into my research.
After a couple of months of figuring things out, by the fall semester I was well into my research.
Chase Van Baalen, my major professor-a gem!

         My PhD research started with purified spinach Rubisco followed by isolating and purifying this enzyme from various microorganisms. The idea behind this work was to figure out why carbon isotope measurements of modern and ancient sediments and rocks were so different from one another. Precambrian stromatolites were thought to be microbially produced structures formed primarily by cyanobacterial primary producers. One question that persisted was whether or not the Rubisco from cyanobacteria produced the same degree of carbon isotope fractionation as the enzyme from higher plants. At the time, Tabita and others thought that the cyanobacterial enzyme had a different structural form from the higher plant Rubsico, although subsequently this was shown not to be the case. The photosynthetic bacteria, Rhodospirillum rubrum, produced two different forms of the enzyme: form I, the high molecular weight form, and form II, a Rubisco with a smaller molecular weight. We wondered whether the different forms might be associated with different enzymatic mechanisms and consequently different isotope fractionations.

          At the time Rubisco isotope research was a very hot field. A group from New Zealand published experiments using soybean enzyme, which showed little effect of bicarbonate concentrations or temperature on fractionation. A team led by Bill Sackett at Texas A&M published work using sorghum Rubisco and measured extremely variable carbon isotope fractionations. We did not find this work compelling given the large differences between the two temperature treatments. Further, Sackett’s group used a cell-free extract, but not purified protein. A French group conducted experiments that produced results having a wide range in values that was completely beyond that known measurements in natural samples. Neither of these groups used Rubisco from taxonomic groups other than higher plants.

         My challenge was to grow microorganisms in large quantities from liters of pure cultures and to purify the Rubisco to homogeneity, i.e., isolating it from all other proteins. The work required that I learn sterile technique and microbial culturing. Once the organisms had been harvested, enzyme purification required column chromatography using size exclusion principles. The location of Rubisco separated by chromatography was assayed by radioactive bicarbonate (14C) uptake measurements. Once the Rubisco was identified, I purified large amounts, milligrams of pure protein, for the isotope experiments. Rubisco was incubated with milligram quantities of the substrate RuBP with an excess of bicarbonate in a buffered solution. When all of the RuBP was used up, each of the 40 experiments was terminated and the product, phosphoglyceric acid (PGA) was purified by crystallization.

         Once the PGA was purified, I proceeded to Pat Parker’s aging “Craig line”—a variation of hand-blown glass tubing modified from Harmon Craig’s original vacuum system. My sample was then pushed into a furnace in which oxygen was added, the PGA was combusted, and CO2 was produced.  The carbon isotope combustion of carbon dioxide was analyzed on a 1960s version Nuclide 6” isotope ratio mass spectrometer (IRMS) complete with now archaic electronic devices and strip chart recorders for recording the isotope compositions. In those days, measurements were completed by hand using rulers and slide rules to calculate the isotope values. Our work showed that different enzyme forms and different metal cofactors resulted in variations in isotope patterns.  We also showed that the enzymatic isotope change between the first product of photosynthesis and carbon dioxide was greater than that measured in vivo with whole plants and cells. My research still provides the foundations of understanding the evolution of photosynthesis on earth, how climate cycles have changed over time, and how plants manage their water budgets.

            My life in Texas continued on two planes—the academic, quiet university life and a rambling, often disorganized personal side. I learned years later that I was known as the “Phantom” at the Marine Science Institute. In the community, I was treated as a responsible nerd. I kept the books and did the ordering for the ice cream business, worked the hotel desk Friday through Sunday, and continued with my eyeglass lens jewelry business selling in local tourist shops. Dedicated work in the lab made the next two years fly by. I was finishing up my dissertation by April 1977, less than three years after beginning grad school. These days a three-year PhD is nearly unheard of. However, I had accomplished more than I had proposed. I defended my work in Austin in mid-May.

         Early on the morning of my defense, professors Parker and Van Baalen came by with fellow student Brian Fry, who was defending his Masters degree that same day, and together in the Institute’s station wagon we drove 150 miles north to Austin. I presented my dissertation work to my committee that included Parker, Van Baalen, Bob Tabita, Jerry Brand (a botanist), and Knut Schmidt Nielson (a famous animal physiologist).  It was a fairly cut and dry exam, over in two hours. We drove straight back to Port Aransas in late afternoon. When I arrived, no one greeted me to hear the good news. Typically, newly minted PhDs are regaled with a party with their friends organized by spouses. The lack of any celebration was one of the first major warning signals that Jack harbored jealousy over the success of my new career. In 6 weeks, we packed up an old trailer, hitched it to the back of a rusted out station wagon and drove north to Washington DC to start the postdoc at the Carnegie’s Geophysical Laboratory.

Monday, December 23, 2019

Merry Iso$$ope Christmas

Proof that Gremlins can and do infect isotope labs
            It was Christmas Eve December 24th. Around the world isotope geochemists, ecologists, and biochemists had put their isotope ratio mass spectrometers and peripheral devices on standby for the holidays. Helium tanks exhaled a collective “sigh,” happy to enjoy an extra few days in their tanks. Furnaces breathed fresh air as they cooled. Copper granules relaxed. Valves in the Isolink were communicating with the Isodat software, planning their annual Christmas bash where they both go wild, hookup, and blink on and off randomly. The turbo pumps were sending out whining calls to the direct drive pumps preparing to undergo their annual Christmas secret Santa giveaways exchanging oils.
            Meanwhile, Isohope, a young graduate student at a Midwestern University, snapped her laptop shut and boarded a bus to visit her family after a late afternoon meeting. Isohope had been trying to measure some traditionally difficult things on her lab’s IRMS facility this fall. She thought it would be easy to measure nitrogen isotopes in tree ring samples—that failed. Then she shifted to measuring nitrogen isotopes in Precambrian rocks 3.8 billion years old—she found only contamination. But Isohope didn’t give up! She tried clumped isotopes in archeological human bones—but they had been too weathered for any useful information. Finally, she selected carbon isotopes in soils. That worked. Sure enough, they all had values between -25.5‰ and -26.5‰. But, and this was a Big But—there wasn’t enough to write a PhD dissertation on soil carbon isotope values. She had one last big idea.
            Earlier that year her fellow grad student, Isodope, had published two papers on his work correcting isotope values of marine carbonates using eleven different equations developed by Isopopes over the past half century. His results apparently appeased the Isotope community’s penchant for small differences in isotope fractionations. But as far as Isohope could determine, Isodope hadn’t really discovered anything. He had just rehashed old science, put it into R Studio and drew an ellipse around it. Isohope wanted more.
            She had spoken to the lab manager, Isocope, a patient, long-suffering individual who listened to students’ complaints, fixed pumps, ordered gases, and in general kept the lab’s Isopope, Isohope, and Isodope happy and “in business”. Isocope looked forward with some trepidation to the holiday, because it seemed that every year the lab was put on standby, Laboratory Gremlins had an informal “takeover” of the instruments, such that come January, none of them wanted to return to work. At the end of the day, Isocope resorted to a stiff snort of Christmas eggnog laced with brandy and put her feet up in front of warm fire.
Valery Terwilliger made friends with them. It helped!
            Meanwhile, the lab’s Isopope wasn’t much happier. Recently NSF and DOE had turned down his proposal for a large format mass spectrometer. His colleagues were starting to call him Isomope instead. In fact at the recent AGU meeting, he’d bad-mouthed all of his colleagues calling them a bunch of Isonopes—out to get him and give him bad reviews. He basically Isomoped around the house, until his wife told him to get his ass in gear and figure out how to reclaim his Isopopedom before he drove everyone mad. Isopope’s wife, Isolope was used to running her own show—and often getting little credit for it.
            Isolope, racing between kids’ soccer schedules and making sure Isopope’s lunch was made, was the real brains behind the lab’s successful operation. Isolope met regularly with Isohope and Isocope to figure out new methods that would give the lab prominence. The three women met that December 24th briefly for a glass of Chardonnay. They hatched a plan for early January. Isohope had a brilliant idea to measure hydrogen isotopes in the metabolites of deep earth microbes that divided only once every 200 years. Isocope thought that she could re-jigger the Isolink to get the sensitivity, while Isolope would do the run around on her husband, Isopope, to make sure the instruments were available and not tied up with contract work.
            First, however, they needed the cooperation of their instrument’s engineers, Isosoap—a squeaky clean, button-downed shirt kind of guy—and Isorope—always figuring out fixes that nearly cost him his job with The Company, because he saved customers money. Isolope knew what to do. She’d sent Isosoap and Isorope each a bottle of Chevas Regal 25 year old Scotch the week before. Isolope had signed Isopope’s name to the gift, puffing up the egos of Isosoap, Isorope and their managers. When a purchase order came in for a specialized part, Isocope, Isohope, and Isolope received the magic bullet to construct the most sensitive, linear, standard-oriented component ever to grace an isotope ratio mass spectrometer.
Seth Newsome's lab uses them for tricky repairs.
            Isomopes and Isonopes had weighed in on Isogeochem voicing their learned opinions that this could “never work—Urey had tried.”  One Isonope cited Slater’s early work on the multi-cluster F---valves that just couldn’t be programmed to do what Isohope hoped. Another Isomope wrote that he’d tried it in the late 1990s with a mercury-adapted part and the results were “all over the place.” Another Isodope wrote and asked the List to send all the papers on how to use an elemental analyzer and a mass spec to measure carbon isotopes in soils—again.
            Isohope’s bus finally reached her hometown of Jefferson, Iowa. It had snowed a few inches, the roads were slick, but her parents met her at the bus station.
            As they drove home, her phone lit up with a text from Isotope Queen—
“Peace be with you and yours. May your mass spectrometers and devices have a happy holiday. Try leaving some raw data for the Gremlins to chew on. Good luck!”

Rounding Third Base and Heading Home

Cards from Franny and Flowers the Rumbles   My daughter Dana is marrying George Goryan on June 25 at our home in Mariposa...