Sabbatical Notes from Bassett Research Institute

I am working on two different projects for my sabbatical, a basic science project (fat cell metabolism) and a history of women in science project (the life and work of pediatric cardiologist Helen Taussig).

Below I've posted entries from my research notebook, which I use to keep track of my plans and activities.

Entry #1
The basic science project I can do locally at the Bassett Research Institute in Cooperstown. Last fall I made contact with Dr. Allan Greene, who is the director of the Research Institute. He was and is incredibly welcoming, and it has been a delight to get to know a whole new set of scientists who are right here in the neighborhood!

Dr. Greene is involved in both clinical and laboratory research, and I decided I'd like to get involved in looking at fat cell metabolism. I'm actually most interested in how a particular naturally produced communication molecule, adenosine, functions in all kinds of cells and since Dr. Greene works with fat cells, they became the model system. This work will give me the chance to develop my skills in cell culture and biochemistry techniques, which I really only know from a textbook point of view right now. Eventually it will help me with other experiments that I plan to run with students back at Hartwick.

After reading some of the background material Dr. Greene gave me to prepare to work in the lab, I realized that this project was going to do a lot more for me than just letting me pursue a long-time interest. I have already learned many new things about diabetes and obesity and the role that fat metabolism plays in both. Among both scientists and the general public, it seems, we make the assumption that fat cells are not very metabolically active but, rather, just passive storage sites. THAT'S NOT TRUE! In fact, they are quite active and play an important regulatory role in metabolism.  

Furthermore, when fat cells are resistant to insulin (as in people with Type II diabetes or even prediabetes) that doesn't just affect the amount of sugar in people's blood–which gets monitored very carefully–but also the amount of fat. That's why people with diabetes have such a high risk of atherosclerosis and heart disease. So I am excited about opening the door to understanding a whole new area of biology that is related to my interests in the cardiovascular system.

My other project, on the Life and Work of Helen Taussig, is progressing more slowly. There are two people who are very important in the story of Taussig's work–Vivien Thomas and Alfred Blalock–and I have to obtain and read both of their biographies carefully, looking for clues about their relationships with Taussig and the working climate at Johns Hopkins that led to the development of the blue baby operations. There also have been two recent films–a documentary and a made-for-TV movie–that tell the story but with minimum attention to Taussig's role. I have to figure out why she is represented so little in these films. I have some ideas about this, but I also have a lot more reading to do before I can draw any conclusions. 

So my travel to the Alan Chesney Archives at Hopkins, where many of the documents from all three of these figures are kept, will have to wait until I get further along. I need to figure out what questions I will  be asking as I review the documents in order to be able to make the most of these trips.

So that's the report for now. More to come. Laura

Footnote: In October, Professor Malloy presented: "Blue Babies and the Birth of Pediatric Cardiology: The Life and Work of Helen Brooke Taussig"

Entry #2
I have spent most of my time in the lab lately learning new techniques. I was trained as a physiologist so I have a lot of experience doing things like measuring blood flow, blood pressure, force development in smooth muscle, and electrical activity in cells. However, while I think a lot about cellular and biochemical mechanisms, most of my work has been done at the tissue and whole organ level of organization. I came to this lab to learn a bit more from the biochemical perspective. My goal is to quantify G proteins, which are proteins imbedded in cell membranes that help translate chemical messages from outside a cell to actions on the inside of a cell. There are excitatory (Gs) and inhibitory (Gi) proteins and in fat cells their relative abundance can have an effect on how much the fat cells break down and release their fat. It appears that G proteins are "down regulated"–that means produced less–when cells are chronically stimulated with adenosine and molecules like it. So I want to repeat the experiment that shows that the amount of inhibitory G proteins goes down when adenosine receptors are stimulated. After I do this, I’ll try to interfere with a nuclear regulatory protein, NFkappa B, that we think is essential in that down regulation process. If when NFkappa B is not produced, G proteins are also not down regulated, we will know that NFkappa B is essential to the down regulation process.

So how will I measure G proteins? I'm practicing a technique that I understand from a textbook and teaching perspective, but that I've never used for my own basic research. I do gel electrophoresis to separate proteins in extracts from fat cells cultured in the presence and absence of adenosine for a couple of days. The electrophoresis works because some proteins are bigger and more negatively charged than others. The electrophoresis cell creates an electric field that is negative on one end and positive on the other. When a gel  is placed in the electric field and loaded with proteins, the biggest and least negative molecules move most slowly while the small and more negatively charged move faster toward the positive end of the cell. So the proteins spread out by size and charge. Then I transfer the proteins to a filter paper (called a "Western blot") and then I use antibodies for the G proteins to attach a luminescent dye to them. That way I can selectively make just the G proteins visible and see how much of them there are. It is really neat when it works, because you actually get to see the marks from tiny amounts of G proteins after going through many, many steps where they are invisible. There are a whole lot of steps in the process, though, so there are also a lot of places where you can make a mistake. The folks here are incredibly helpful and have been teaching me a great deal about how to troubleshoot this technique. I am really enjoying the work and hope that we get some interesting results. Laura

Entry #3
I took some vacation time and went out to Otego to attend a pottery workshop conducted by Elizabeth Nields. You might recall that Elizabeth is an artist in residence at Hartwick and she is also on the faculty at SUNY Oneonta. It was a wonderful experience. I improved my skills as a potter and learned a great deal about ceramics as an art form from Elizabeth as well as the other potters who were there. She has a show every year in August and my work was well received at this year's exhibition. What I love about making pottery is that it is so different from the way of thinking that I use as a scientist and a teacher. Almost all of my scientific work depends extensively on language--on verbal and written skills. Language is not as essential to making pottery, but three-dimensional thinking and tactile experience are. This lets me get deeply engaged in something completely different--so I get to exercise a less used part of my brain! (I just can't help doing this biologist's interpretation of the experience!) It feels great and it a wonderful way to rekindle my energies for my other work.

I also continued to develop my biochemistry lab skills at Bassett Hospital   Because we will use a virus to halt the expression of the fat cell proteins of interest, I am learning techniques that will help me to handle the virus safely.  This is a cold virus that has been disabled, so it is not particularly infectious or dangerous, but one must still take precautions in handling it very carefully, since it has been genetically modified. We only handle the virus under sterile conditions in a laminar flow hood in a restricted area. I have been developing techniques that reduce the number of steps required to prepare fat cells once we have treated them with the virus. If we can detect the proteins we want to evaluate (the G proteins) from whole cell preparations instead of cell membrane extracts, it will reduce the number of times the virus needs to be transferred from one tube to another and thus improve our safety precautions.  So that's what I will continue to work out.

I've also managed to do a bit of background work on my biographical project on Helen Taussig. I find it really interesting that recently two movies--one made for cable TV and another for public television--came out on the Blue Baby Operations. In both movies, Taussig's role in developing the idea has been minimized because she didn't play as active a role in developing the technique. I find this interesting because most of the time in science the technical side of the scholarship is minimized. This might be the difference, however, in telling the story from the point of view of a surgeon (where technique is a very important part of the enterprise and technical prowess a big part of the disciplinary culture) versus the points of view of a cardiologist or a scientist. So I have to read more about these disciplines and their cultures as well as the ways in which the films were made in order to understand how this piece will fit with how we view Taussig's work today. 

Well, that's all for now. I'll send along another update soon. Laura

Entry #4
Things are going really well in the lab. I have gotten the electrophoresis techniques down and am still working on the tissue preparation. Results from my first two experiments show that my cell membrane preparations (which I didn't think I was so good at) worked just fine and I was able to identify both of the G protein inhibitory subunits, even though they are very hard to separate.  However, I remain concerned about the whole cell preparation. I am hopeful that I will still be able to see the G proteins in the whole cell prep. The potential problem is that with all the other cell proteins left in the whole cell prep, the G proteins could be obscured--that is, the signal muddied so that I could not separate the two different proteins from each other. My first results were so good, however, that I  remain optimistic. I very much want this to work, because the new technique would be easier and, more importantly, safer. I'm going to keep this short this time, as I've spent so much time in the lab that I haven't worked on my other project and I sill have lots of work to do.

I'll be in touch again soon. Laura

Entry #5
I am afraid I don't have a lot to report for October. The very promising results that I started out with seem to have dried up. I am rechecking basic techniques but can't seem to identify what may have changed in my technique. It seems that the last step in the process to develop the gels after I have run them just is not working. I have started to take the protocol apart systematically to see if I can identify what is going wrong. Meanwhile, I also got some frustrating news about my other project – the archive where I planned to work will be closed until some time at the end of November, so I will not be able to make the trip for that project until sometime at the very end of my sabbatical. Sometimes scholarship has its low points and it seems I've hit one this month. Hopefully I'll have some more positive news next time around. Laura