Greetings from New Zealand!

Sorry to have been out of touch but having some technical difficulties.  My computer was great for 6 weeks but now seems a bit tempermental.  The joys( not) of airport internet access.  While in NZ we’ve travelled along steep winding  roads with BEAUTIFUL and awesome scenery.  Mountains green, brown and gray.  We went out to a gannet breeding colony and saw the chicks feeding by putting theirbeaks waaay down a parent throat.  There were at least 100 birds right in front of us not carry whether we watched them as long as we stayed about 5 ft away.  It was very interesting actually see bird behavior I’d only read about (or seen on videos) before.  The fact that the birds were territorial was obvious.  Each nest, more like a flattened mound, was defended by an adult.  The babies were really late juveniles, the same size as their parents but with a mixture of white, brown and gray feathers rather that the white and creamy yellow headed adults.  Soon the young will go one direction to feed and grow for 3-5 years before returning to the same place to breed.  The adults will go another direction to feed until next breeding season.  The separation probably to reduce competition between the two subpopulations.

We went out on a boat and spent 20 minutes watching very rare Hector dolphins – a pod with juveniles babbies and mothers.  They came right up to an even under the pontoon boat.  We also saw four different species of water birds called cormorants as well as the NZ pigeons (not like the ones a Ball Hall) and NZ robins which are smaller than our robins but hop around like the robin we know.  We also tasted some great NZ wines.

This country seems to do a great job of balanced land and water use.  They say they’re interested in sustainable use of their resources and what I’ve seen is consistent with that.  They plant trees to harvest and leaveother areas wild and untouched.  They build resorts and golf courses but in the same area are dedicated to remove all the human-introduced predators so that one day the endangered kiwi (NZ’s iconic bird – not the fruit) can be re-introduced.  Sheep and cattle graze on brown grass on what would seem unscalable inclines.  Most of the land appears untouched and the shorelines are breathtaking one after another after another.  If you like seafood – fish and mussels and oysters and scallops – and I do – this is a wonderous place! It was a great 12 days relaxing, studying the environment and eating fabulous food.

On the ferry between the north and south island the water was every color possible from sea green to cobalt blue to gray!  Brilliant!  Gotta go only a few mintes left.   Actually I got to edit at a free site in the Sydney airport.

Today we’re flying to Sydney and then to Honolulu.  Looong flight but expecting more adventures.

Will let you know more soon, I hope!

This is my last week in the lab in Sydney.  On Monday I practiced a technique called allelic discrimination.  As with the luciferase assay I described before this technique depends upon a special machine which both provides the data and analyzes the results.  We examined a series of DNA samples from people who serve as controls for the study of schizophrenia that is the major focus of this lab.  The technique is based on another technique called PCR (polymerase chain reaction) by which you can take a small sample of DNA and make many reliable copies.  I’m fairly confident you understand the facility of this technique in forensics.  Using this technique, DNA from a tiny blood sample found at a crime scene can be amplified (so there’s enough to test) and then compared with the DNA of a possible suspect.  I think you’ve probably also learned that humans have two copies of each gene (one per chromosome).  In a simple example often used in basic genetics classes there are two alleles (forms) of the gene for an enzyme that when non-functional leads to a person lacking any pigmentation in his/her skin, a condition known as albinism.  What we were looking at was much simpler, a difference of one nucleotide (a C vs. a T) at a specific site within the gene for the estrogen receptor (ER alpha) that I have written about before.  When one nucleotide is different it is called a SNP (single nucleotide polymorphism).  Within a DNA sample from any person, there will only be two copies of this sequence; so there are three possible genotypes CC, CT/TC or TT.  These two copies of that SNP are not enough DNA to compare; we can’t “see” it with any technique or technology.  The DNA is amplified using PCR.  The region we’re interested in is selectively copied because we provide primers, pieces of DNA that will match the two ends of the sequence bracketing the SNP.   In PCR the DNA sample is heated so the two parts of the double helix will come apart and become single stranded.   Then the temperature is lowered to allow the primers to attach and the heat stable DNA polymerase makes copies.  There are multiple cycles so that many copies can be made. The primer DNA provided by the scientist (me) is complementary to the DNA near the region of interest so it will match up with that part of the DNA in the sample.  Now here’s the discrimination part.  We also provide probes; one that is complementary to one allele and another that is complementary to the other allele within the bracket of the primers.   Each of these probes has another component at each of its ends; one colored and another that keeps the color from being “seen” by the machine, this is called a quencher.  Let’s say the probe for C has a red tag and the probe for T has a blue tag.  OK, if the genomic DNA matches exactly (is complementary along its entire length) with the probe, then the DNA copying enzyme running along the genomic DNA making a copy will bump into the probe and chew it up (digest is a more scientific term) thus freeing the colored tag from its quencher.  If the match is not exact, no chewing, quencher stays with the colored tag, no color.  OK so if the genomic DNA has C at the SNP spot, the red-carrrying probe will match and the red marker will be seen by the machine.  Alternately if the genomic DNA has T, by the same mechanism the machine will see blue.  We put the same primers, probes, enzymes in all the wells of our sample plate.  We then put different DNA samples in each well.  Each well contains only 5 microliters of fluid = 0.005 ml = 0.000005 L!  Think of the size of a 2L bottle of soft drinks divided many many times.  I worked with 20 samples.  We then program the software of the machine to read our plate, put the plate in and start the process.  First PCR copies each DNA sample many many times.  During the copying red tags will be released if C is present in the SNP site and blue tags if T is present and red and blue tags if one chromosome has C and the other has T.  Then the machine reads the amount of red and blue in each sample well and reports the results.  In my sample there were five CC, eight CT and seven TT for a genotype frequency of 25%:40%:35% and an allelic frequency of 45% C and 55% T.  The entire experiment took about two hours including just over one hour of the machine working without my doing anything. 

Such a machine is likely beyond the financial resources of a small college like Keuka.  It is used here not only by the members of Cyndi Shannon-Weickert’s lab but also by other researchers studying the brain at the Prince of Wales Medical Research Institute.  My challenge is to conceive of ways that Keuka students can study some of the same concepts with simpler technology.  We can (and already do) perform PCR.  I need to learn how to take those products and discriminate between them using a slightly less high tech methodology.  And then constructively add new experiences to what is already part of the natural science curriculum. 

Yesterday (Wednesday) Cyndi made some time to work with me “at the bench.”  We each took a plasmid (I wrote about plasmids previously) with a human gene insert, I had the human glutocorticoid receptor, and treated it with a selected restriction enzyme to linearize it.  It was incubated in a water bath overnight.  Today we will “run” our pieces of DNA out on a gel.  This morning I learned how to make agarose gel solution (using a microwave) and to pour a gel for electrophoresis.  The most challenging part of this is putting tape around the frame just so before pouring the liquid gel so that it doesn’t leak.  The idea is that we’ll put samples of our DNA into little wells in the solidified, jello-like matrix of agarose and then electrophoresis will allow us to determine the different weights/lengths of the pieces of DNA we’ve produced.  This is actually a very straightforward and common technique, performed by students in biochem or molecular bio labs.  But, I’ve never done it before!   Oh and the first gel we tried to make leaked all over the lab bench!  So I got the practice of starting again, alone and I have made a “perfect” (at least it solidified without leaking) gel by carefully considering the taping technique.   I’ll report more in my next message. 

It has been very interesting spending the last five weeks in a working research lab.  There are four post-docs, two technicians, an undergraduate student, a lab supervisor and an executive assistant as well as Cyndi who is the principle investigator (as well as many other titles and responsibilities).   I want to remind you that Cyndi is a Keuka graduate from the mid 1980s with majors in both biology and psychology.  She was well-prepared at Keuka and with determination and desire furthered her education and rose high in her profession.  From my other messages you can tell that I’ve been working with everything from sectioned tissue to tissue culture to cortisol to DNA.  When we’ve worked with the microscope, I’ve had experience to contribute.  We’ve also read and discussed articles at journal clubs at least once a week.  I’ve learned a great deal about neurobiology and I’ve been able to contribute my broader knowledge of cell, developmental and molecular biology in a number of non-neuronal systems.  The work here also includes the behavioral aspects of neurobiology.  Cyndi is responsible for seeing the big picture and charting the direction of research as she and her colleagues strive to understand what “goes wrong” in schizophrenia and how it might be prevented or more effectively treated.   I have been stimulated and challenged to both expand and focus my thinking as I strive to understand and to provide a semi-outsider’s insight.  Everyone has been very friendly and welcoming; generous with their time and encouragement.   This is the way they interact with each other and they have welcomed me into the group.  We have worked and laughed and learned together.  (As they say here) brilliant!

Our trip to Australia has provided the opportunity to feed another of my obsessions, birdwatching. Birdwatching is a hobby wherein one attempts to locate, identify and observe different species of birds. It is a bit like stamp collecting in that you collect, on a life list, all the birds you’ve been able to accurately identify. I am a lister but that’s not my passion. I like to study information about a place I’ll be visiting and set my search image for the birds I might see. In addition to a spectrum of those possible, I usually identify a dream bird; a spectacular looking bird that will probably be difficult to find and will be satisfying when seen. Identifying a new bird is a rush, possibly similar to scoring a goal in a soccer game. It is a wonderful challenge; spot a movement, get your binoculars on it; check what you see against the birds you know to expect. Or collecting the identifying features like color and size and bill shape and then looking quickly through a book to try to find something that looks like what you saw and hopefully having a chance to look again. Australian birds include numerous spectacularly large and colorful birds including the following that I have seen: Rainbow Lorikeets, Australian King Parrots, Crimson Rosellas and Sulfur-crested Cockatoos. Each of theseidentifications was a fantastic experience. Should I say awesome? Sulfur-crested Cockatoos are large white parrots with a tuft of lemon-colored feathers on the crest of their heads. They also have a blaze of yellow under their spread wings. We first saw them in the Botanic Gardens near Sydney Harbor. People feed them bread and get them to perch on their shoulders. I find this practice disturbing but more on that later.

This past weekend we saw Sulfur-crested Cockatoos “in the wild” in the Blue Mountains. You can imagine how conspicuous a white bird was against forests of lush green trees. On Sunday we were walking in the Blue Mountains and came upon two Crimson Rosellas, large parrots with red heads and upper bodies, blue wings and tails, sitting just in front of us perched in a tree. The four of us were the only people there.

It was a heart stopping moment! Serendipity rewards the prepared mind. Mary and Cyndi found them but I knew who they were! Later in the day we saw the King Parrots, at a greater distance than the rosellas but we were easily able to identify them. The females are mostly bright green while the males are green with orange upper bodies. I didn’t see my dreambird for the trip to the Blue Mountains, a male superb fairy wren. He is a tiny bird mostly black with iridescent sky blue markings on his head, face and wings. Several people were kind enough to tell us where to look but we were not lucky. For me, a dream bird is a talisman, something to look and hope for. I want to see it (well I want to see any birds I can find) but I look carefully for it and I see many expected and unexpected birds as a by product of this focused observation. Oh, and the Rainbow Lorikeets we’ve seen just in walking around Randwick (the suburb of Sydney where we’re staying) including around a dozen at a time at a birdfeeder that is on my walk from the flat (apartment) to the lab every morning and evening. These guys live up to their name. The impression they give in flight is mostly bright green but at rest you can see they have blue heads, green wings and backs, red chests and scattered patches of bright yellow. A truly spectacular bird! As I’m writing this I heard a lorikeet and looked out to find one eating from blossoms right outside the lab window! In addition to the spectacularly colored birds we’ve seen many birds that are mostly black with white markings, Magpies, Magpie larks, Pied Currawongs, New Holland Honeyeaters and Red Wattlebirds.

Another important byproduct of this searching for birds is that it focuses your attention on the physical and natural environment. Watching birds over the years has helped me to develop deeper insight into ecology and adaptation than I ever learned in a class or from a book. BUT the book and classes gave me the terms and the framework that helped my accumulating experience of nature lead to understanding. The plants in Sydney remind me of my experiences in south Florida; palm trees, flowering trees and bushes. The profusion of flowering plants here is arresting and there are many different types of birds adapted to eating from those flowers directly or eating the insects that gather at the flowers or eating the fruits that the flowers ultimately become. You may be familiar with hummingbirds that we see in the US. No hummers here but the honeyeaters and their relatives do the nectar harvesting as well as eating insects. The parrots also seem to favor the flowers. This morning (Tuesday) there were at least 10 Rainbow Lorikeets in a flowering tree outside the window near my desk in the lab. The tree has just come into full flower. We noticed the lorikeets were not at the feeders I mentioned. Possibly they’ve switched to this tree while the flowers are just right for their taste. This reverie reminds me of another amazing experience we’ve had here. Observation of 100s of flying foxes both at a distance and up close. Flying foxes are a type of very large bat. Now, before you shiver and say no thanks I’ll skip this part. Let me tell you a bit about mammalian evolution. The earliest mammals were insect eaters. They were around at the same time as the dinosaurs. The mammals ate insects while the dinosaurs ate plants and other animals (mostly non-insects). Thus there was little competition. One explanation for the evolution of bats was the large numbers of available insects at night. In time the bats diversified and some specialized to feed on fruit that some of the insects were feeding on. The evolution of flowering (and fruit making) plants came along as the bats were diversifying. [I ask the indulgence of those who read and recognize inaccuracies produced by my over-simplification of this bit of evolutionary history]. Anyway, compared to the more familiar bats, the fruit eaters have large eyes and ears and a longer snout correlated to feeding in the daylight or twilight at the beginning and end of the day. Thus the name flying foxes. I had seen videos of flying foxes but seeing them “in the flesh” was really exciting. We went out to dinner at a restaurant on the water at a place called Wollomoloo (there’s music in some of the place names here) and at dusk, while we were having a glass of wine, we saw large black bird-like shapes along the horizon on the other side of the water. “Those are the fox bats” Cyndi said. Later in our visit to Sydney we spent time at the Botanic Gardens which is also on the water and discovered that the bats we saw from the restaurant had been going to roost in the trees of the Botanic Gardens. The trees in the gardens are full of flying foxes hanging upside down from the branches! The common name of this type is Grey-headed Flying-foxes. To a naturalist interested in animals this profusion of bats would seem a good thing; an opportunity to study them up close. We spent some time watching them interact. Screeching at each other and changing position; maybe based on dominance? Very interesting. But as we strolled through the gardens we became aware of how many bats were present; at least multiple hundreds, perhaps as many as a thousand. A handout and a conversation with a volunteer revealed that this was way too much of a good thing. Apparently the natural habitat of these bats elsewhere in New South Wales had been disturbed, possibly by development for hotels or other living spaces for people so the bats were forced to move and discovered the Botanic Gardens. Probably they were spread over a much greater area previously but now they are highly concentrated in one small area. Their presence has already destroyed some very old trees. So according to a handout I picked up “the Royal Botanic Gardens has been given a licence by the New South Wales National Parks and Wildlife Service to use non-harmful methods to discourage the bats from roosting” there.

Studying animals as a hobby as well as a profession makes me feel close to nature and makes me conscious of the effects we humans have on the natural environment. Feeding bread to birds in the Botanic Gardens leads to exciting experiences and photographs for the people providing the bread. However, feeding this way is unnatural behavior for the birds and may make them dependent, result in crowding that can spread disease amongst the birds and result in pest birds that might even have to be destroyed! The Blue Mountain region includes a national park and is similar to the Adirondacks in New York in that people’s homes and recreational areas are scattered around protected natural areas. There’s a not so positive tension between our love of other animals and the beauty of a seaside or lakeside view and how our actions may effect those animals, that seaside or lake shore. I think it is important to think about as well as feel our connection to nature.

We visited the Hunter Valley Wine region two Saturdays ago.  I very much enjoy different types of wine.  I particularly like planning a meal to prepare and then pairing it with the right wine.  Living in the Finger Lakes I’m surrounded by opportunities of different wines to experience.  Also, I’ve sampled wines from Australia from liquor stores in Rochester and Ithaca and I liked some of those.  But there are wines that don’t get exported because they are only produced in small quantities or someone thinks they won’t be popular in the United States.  It was those “local” wines that we were most interested in trying.  We did some reading before we left Sydney and also found that there were some wineries that also had their own olive trees and press olive oil for sale. The Hunter Valley is a drive of about 2 ½ hours from Sydney.  It is different from the Finger Lakes wine country for many reasons.  The climate is warmer and there aren’t the familiar lakes around which our wineries are clustered.  As we were driving from winery to winery the terrain seemed too flat for vineyards (based on my expectations from the Finger Lakes) but Mary and I decided it was more like the Napa Valley in California which we visited about twenty years ago.  Anyone who has wanted to visit a winery on Seneca and then another winery on the far side of Cayuga can identify with the pleasure it was not to have to go around lakes!  Australian wineries call their tasting rooms cellar doors.  But, as in the Finger Lakes, those that pour the wine are friendly and knowledgeable.  There are two white grape varieties that are characteristic of the Hunter Valley Semillion and Verdelho.  I’ve had Semillion Chardonnay blends from Australia at home and once tried a Vedelho.  So I was particularly intrigued with tasting a wider selection of these at the wineries.  We visited several wineries and tasted many wines that were either 100% Semillion or Verhdelho or blends.  I must say that in some cases I couldn’t distinguish between the two.  They both taste citrusy – lemony or limey, not sweet but, to my palate without much character.      In general I didn’t find a white that I liked more that a Finger Lakes Dry Riesling or, my very favorite, Sauvignon Blanc from Marlborough in New Zealand.   Many regions of Australia, including Hunter Valley are known for the red wine grape Shiraz.  Shiraz has many enthusiasts and I like to try new things.  But, I have not been very impressed by the ones available in US liquor stores, too fruity for me.  I like dry peppery reds, particularly Cabernet Franc which grows well in the Finger Lakes.   I liked the local Hunter Valley Shiraz more than those that are marketed in the US but I liked Shiraz best when blended with other grapes. We decided we would buy some wine in the Hunter.  Just enough to consume here since it is quite a process (and quite expensive) to ship it home.  We did have one Verdelho (at Hungerford Hills) that we liked enough to buy.  The best white, to my palate, that we tasted was a blend of Chardonnay, Semillion and Vedelho (at Bimbagen).  It was very good with the pumpkin and bean soup that we made in our flat.  We also bought some Merlot (again from Hungerford Hills) which we enjoyed with lamb chops, brown rice and a green salad with blue cheese.   So even while in Australia I got the chance to select wines from our mini-cellar for meals I cooked.  What fun!  Oh and we bought some great olive oil that we will be bringing home!

I successfully replicated the experiment!  As I described in my last post, a machine measured the light produced by fruitfly luciferase, compared it to the light produced by sea pansy luciferase and the ratio of the products increased as a function of the estradiol I used as a stimulus.  Those of you that know about such things will know I’m happy about the small error bars.  It suggests I also did a good job in reproducing the same volumes in the sample wells.  I’m a bit self-conscious about my micropipetting technique.  (I have a graph to show you but copy and paste from Excel doesn’t seem to work).

This week’s work will be with developing facility with a technique that measures the amount of cortisol (another hormone) in saliva samples.  Those of you that know about the project that Camille Fontaine developed last year may find this familiar.  More later.

Yesterday, I again changed the media on my cells and then added in the mixture that, hopefully will allow me to measure the response of the estrogen response element (DNA) to different amounts of estrogen.   Picture this: my cells are growing on the bottom surface of a little well.  Layered over the cells is a pool of pink medium that will provide the nutrients that the cells need to continue to grow.  I prepared a mixture of 4 substances that when added to the cell cultures will cause specific pieces of DNA to enter the cells (scientists say transfect).  First I put a small amount of medium (it looks like water) into a tube.  Then I added a small amount of liquid that contained pieces of DNA with the estrogen response element (ERE) next to the gene for an enzyme called luciferase from fireflies. Next, I added another liquid that contained other pieces of DNA that with the gene for luciferase from a sea pansy (that’s a soft coral for those who know invertebrate animals).  Finally, I added lipofectamine.  The idea is that the lipofectamine (which is molecularly similar to the lipid molecules in the cell membrane) will help the bits of DNA (called plasmids) across the cell membrane of my cells.  I have practiced genetic engineering!  I returned my culture plate to the 37⁰ C (98⁰ F) incubator.  The cells, plasmids & lipofectamine “cooked” together overnight and we assume tranfection has occurred. 

==== ERE ERE ERE firefly LUCIFERASE GENE ====== (really in a circle) 

==== sea pansy luciferase gene === (also in a circle) 

Today, I again changed the media on my cells and this time I added different concentrations of estradiol to 6 sets of 3 wells each (0, 0.01, 0.05, 0.1, 0.5, and 1.0 nM) and then returned the dish to the incubator.  Tomorrow will be the moment of truth.  The way this technique works is that:  estrogen binds to the ERE and turns on the nearby luciferase gene.   Then the cell makes mRNA and then luciferase protein.  Later we’ll provide a substance (scientists say substrate) that luciferase will convert into another substance that makes light.  And we’ll measure the amount of light which will tell us how much luciferase was made.  My hypothesis is that the lower concentrations of estrogen will correspond to lower amounts of light.  By the way, remember this is from a firefly (or lightening bug).  The chemical reaction is how fireflies make light!   The reason that the sea pansy (Renilla) luciferase plasmid is added is as a control.  Tomorrow we’ll use a machine that adds the substrate and measures the amount of light.  The machine will first measure firefly-luciferase-produced light and then adds another liquid (Stop @ Glo) that stops the firefly reaction and adds the substrate for the sea pansy luciferase reaction.  Then, the machine will measure the light from the second reaction.  We will have a measure of light from both luciferases in each well and the machine will report a ratio.  This provides an internal control so we can have more confidence in our numbers.   

It is very different being on the student side of the bench; worrying that I won’t do everything perfectly and so contaminate my cultures or otherwise stop the experiment prematurely.    My supervisor is very patient and very clear and allows me to run through what I am to do mentally before I have to do it with my hands.  She’s a very good teacher.  She noticed some things that I have trouble with, like remembering the next step or how much to put in, so she taped the steps on the outside of the hood so I can see them easily from where I’m working.  And, we’ve modified the standard procedure (biologists say protocol) to a way that is easier for me to accomplish without mistakes.   My hands shake with a long pipette but with shorter ones I’m doing fine.  It’s a longer process but has contributed to my success.  I can emphasize more than ever with my students when I ask them to do something unfamiliar.   My cells show no sign of contamination!  I seem to be getting better at working with very small quantities and with great care to avoid contamination.  I enjoy the feeling of a challenge met.  We’ll see if I can get the expected curve from the machine tomorrow. 

For you to understand the experiment I’m working on this week I need to provide some orientation to molecular genetics. Most everyone understands that genes are responsible for the characteristics we have. For example there are three possibilities for human hair texture; curly, wavy or straight. What is less well-know by non- or beginning biologists is that some genes are only turned on sometimes (biologists say expressed). For example we humans make a different type of hemoglobin (the protein that carries oxygen to individual cells) while developing in the uterus and getting oxygen indirectly from our mother’s blood than when we’re out here breathing air for ourselves. Thus there’s a mechanism that turns the gene for the in utero type off and the born-human type on near birth. Gene expression is regulated. Most everyone knows that genes are made of the chemical DNA and that the sequence of the famous letters A, T, G and C somehow leads to the characteristic like hair texture or type of hemoglobin (HB). The two types of HB are specified by different genes. The letter sequence of the DNA will be similar, after all both types of protein need to carry oxygen, but somewhat different. Here’s the important part of this story: DNA sequence is copied (transcribed) into mRNA and then the mRNA sequence is used as a pattern (translated) to make a sequence of amino acids and that’s the protein. Thus you can measure gene expression either by determining the amount or location of mRNA of a specific sequence or of a protein of a particular type. So in my immunofluorescence experiment I used the fact that the protein GFAP is expressed in astrocytes but not neurons to identify astrocytes. I had hoped to show that estrogen receptor alpha is expressed in neurons and not astrocytes but . . . One of the areas of biology that I find very interesting is what is called cell signaling. An example of a cell signal, that most everyone knows about are hormones. These molecules are released into the blood by glands and have effects on target cells. In order for a cell to be a target cell it must contain another molecule called a receptor; a protein that specifically sticks (biologists say binds) to that hormone. By the way, this specific binding stuff is a key molecular mechanism is biology. Antibodies bind to antigens; enzymes bind to substrates; signals bind to receptors; transcription factors bind to promoters (regions of DNA involved in beginning the production of specific RNAs). So cells that respond to the hormone signal estrogen contain estrogen receptors.One common mechanism of hormone action is to turn certain specific genes on (or off). So the expression of those specific genes is regulated by estrogen. Essentially it works like this: the estrogen receptors (ER) are present in the cytoplasm of estrogen-sensitive cells, estrogen (E₂ = estradiol, major chemical component of estrogen) diffuses from the blood into the cell and binds to two receptors so they form a dimer. The dimers move into the nucleus and bind to specific sections of the DNA called EREs (estrogen-response elements) turning the nearby genes on. More specifically this binding attracts (biologists say recruits) proteins (transcription factors) and the enzyme RNA polymerase so that mRNA is made (transcribed) from the specific gene. The mRNA moves out of the nucleus and is translated into a protein in the cytoplasm. Many proteins in many different cells are made under the direction of genes that are estrogen- sensitive. We could specifically look for those proteins in response to estrogen treatment. But we’re going to do something different to detect estrogen signaling by measuring the amount of light produced. More about the way that works next time. I’m going to use cultured cells to perform my study. That doesn’t mean that my cells are particularly musical or artistic. It means that the cells I’m going to study are growing in a dish; isolated from an animal. Many of us are interested in how things work in people or animals or even plants. Why would we want to study cells growing in a dish? The answer is that we can more easily control the amounts and types of molecules and other environmental influences (pH, temperature, number of cells) in a cell culture. The cells I’m using are Chinese hamster ovary (CHO) cells. Scientists have been working with these cells for many years. Originally someone isolated cells from the ovary of a Chinese hamster and figured out what mixture of water, ions, nutrients and so forth would allow the cells to grow in a dish. Now one buys the cells from a catalog and we know what medium to use (also purchased from a catalog). My mentor in Dr. Shannon Weickert’s lab is Dr. Jenny Wong. She’s an expert on the type of study I’m doing. In fact, I’m replicating something she’s done before. In this way I can learn the technique and know what the outcome “should” be. If I don’t get the same results either I’ve done something wrong or something else was not controlled as expected. Yesterday, I took a culture bottle on which CHO cells were happily growing and split them. I transferred some into another bottle, to keep the culture going and I transferred some into a multi-welled plate, a clear plastic dish with 48 little chambers and a top that covers all the chambers. For my experiment, I plated cells in 18 chambers. Friday I’m going to test the effects of six different concentrations of estrogen in triplicate. As anyone who’s done any kind of experiment or followed a recipe knows, there are a number of steps that you need to perform in a specific order. What is particularly challenging about this experiment is that everything must be kept completely sterile! We’re all cautioned to sneeze into a tissue and wash our hands. Bacteria and fungal spores are everywhere! We want to avoid them so we don’t get sick. I want to keep them out of my cultures because I want to measure the behavior of CHO cells and not CHO cells plus an unknown number of different types of bacteria and fungi. So we work in a special box (called a hood) that is sterilized with ultraviolet light before we start. The air flow is also controlled so that when the fan is on, air flows out, never in. We also spray the hood and any glass bottles we put in the hood with 70% alcohol. We wear gloves and even spray our gloves with 70% alcohol as well. All the plastic dishes and pipettes (special droppers that allow us to precisely measure the amount of liquid we use) are wrapped until we use them. We work with our arms inside the hood and we are careful never to touch, even with gloves, anything that will come into contact with our cells. I was glad to have Jenny there to stop me before I made an unconscious mistake. Learning the steps and getting your hands to do just exactly what they must do is very difficult. I understand intellectually what must be done but to actually do it effectively requires careful concentration and practice. It’s a little bit like that trick when you try to pat your nose and rub your stomach at the same time; only more so. I successfully platted my cells and observing them today showed no evidence of contamination. Today I changed the media. To change the medium, I had to draw the fluid out of each well, add and then draw off saline twice to get all the old medium without disturbing the cells and then add in new medium. My hands were shaking and I had to change pipettes because I made unapproved contacts. But, I don’t think I introduced any contamination. The culture medium we used to start the little cultures had fetal calf serum in it and we need to grow the cells for a day without that because it has estrogen in it and remember we want to control the amount of estrogen. Check back again. Transfection tomorrow, estrogen treatment on Thursday and looking for that revealing light on Friday.

I just finished looking at the slides from my second attempt at double immunofluoresence. It worked but not perfectly. Remember I wanted to stain two different proteins? For technical reasons we changed the proteins we targeted. We set out to stain ERa as before but the second protein was GFAP. A marker protein for the non-neuron cells in the brain called the glia. My GFAP staining (red) is beautiful! The type of glia I stained are called astrocytes because they look like stars. The fluor is called Texas red; it’s the color of a little-red-wagon or a classic red tulip. I’m going to learn how to take a picture with the very very fancy microscope they have here so I can show you in the future. This scope is so automated you don’t even change the objective lens by hand; you push a button! It feels a little like I imagine the astronomers at NASA feel with they operate a Mars rover! OK so we celebrate the bright red GFAP staining. Unfortunately, the mellow green (like a watermelon pickle) that should be in the cytoplasm or nuclei of neurons is in the same place as the red! Oops! We had something called cross-reactivity because we didn’t wash the slides separately. The secondary antibody (see my previous entry about the immuno technique) that supposed to latch on to the primary antibody for ERa seems to have connected to the primary antibody for GFAP instead. This is not as stupid a mistake as it sounds we had reason to suppose that we wouldn’t didn’t need to worry but more care – more steps – more time – probably would have yielded more satisfactory results. These have been two good half weeks in the lab. I’ve been working in an area of technique that is similar to one that I’ve practiced before and successfully guided students to perform. But this version is trickier because it is more complicated. More things can go wrong so you need to exercise even greater care to control more variables and eliminate more sources of experimental error. If I had more time I could repeat the experiment again and I think I would get it right this time. The researcher who worked with me and taught me this technique, Debora Rothman, will be continuing with the experiment because it is important to the work of this lab that the presence of ERa in the nuclei of brain neurons be confirmed with this technique. Next week I’ll be moving on to another area of Dr. Shannon Weickert’s lab. I will be working with another member of the lab to culture, transfect (insert pieces of DNA) and study the response of cells to a stimulus using light produced by a firefly gene as the means of measuring response. It’s a really neat technique that I understand in theory. I’m anxious to see how it works in practice. But more about that next time.

At Keuka we perform immunohistochemistry in the Cell and Developmental Biology course that I teach each fall semester (taken by sophomore biology majors). We look for one protein at a time and we look for proteins that we know will be there. In our lab the technique is very successful in allowing students to practice the technique and get satisfying results. Some students perform a second experiment with thin slices (we call them sections) of mouse brains of three different types. One type has a mutation in the gene for a protein called dysbindin, which has been implicated in a susceptibility gene for schizophrenia in humans. The other two types of mice have either one normal + one mutant (called heterozygotes) or two normal dysbindin genes. Keuka students are asking the question does the dysbindin genotype have an effect on the presence or amount of another protein of interest. I originally learned this technique from Dr. Cynthia Shannon Weickert (who graduated from Keuka as a biology major in 1987) in whose lab I am currently working.

Here’s what happened when I tried to solve an experimental problem and to practice a more complicated version of an immunocytochemistry technique.

Thursday and Friday of last week I performed an experiment designed to demonstrate that there are estrogen receptors within the neurons found in the brain of monkeys. I only made one procedural mistake and that mistake shouldn’t have eliminated the results. But we will soon try, try again.

I am trying to use a technique called double immunofluorescence microscopy to visualize two proteins NeuN and ER. [Biologist’s use lots of abbreviations. I’ll try to make them clear when necessary]. Neu N is found only in the nucleus on neurons and is therefore a marker protein that identifies specific spots on a slide as the nucleus of a neuron. ERis estrogen receptor alpha; the protein to which the hormone estrogen binds in order to have its effect. We want to know if (well, confirm that) brain neurons have these proteins so we’d know that these cells have the ability to respond to the chemical signal estrogen. When biologists look for specific proteins in cells they often use immmuno techniques to find them. Immuno means that antibodies are used. You probably know that animals produce antibodies to fight off disease-causing organisms like bacteria or viruses. When we get a vaccination we are training our bodies to produce antibodies specific to a particular bacterium or virus. What you may not know is that scientists use animals as factories to produce antibodies to specific proteins so that they can be used in laboratories in many ways including the technique I was trying to practice last week. So, if we use an antibody to NeuN and another antibody to ER those antibodies will attach to the proteins in a thin slice of tissue. Great! Unfortunately we can’t see the antibodies. This is where the fluorescence part of the technique comes in. A fluorescent molecule emits colored light when it is exposed to light. So, if we buy an antibody to one protein with a red fluor covalently bonded to it and an antibody to another protein with a green fluor we can do a double antibody technique on the same tissue and see if the proteins of interest are colocalized. Got it? Good. But it’s more complicated than that.

Antibodies to specific proteins are very expensive. I estimate the amount I used for my one (failed) experiment cost at least $200. The technique that as usually used attaches another antibody to the primary antibody and that secondary antibody is the one with the fluor attached. The primary antibodies to NeuN and ER were made in mice and the secondary antibody was made in donkey and can recognize any mouse protein including the antibodies to NeuN and ERcx.

Y (secondary antibody to mice proteins – made in donkey
Y (primary antibody to NeuN – made in mouse)
NeuN protein

Ok. Do you see a possible problem here? Both my antibodies are made in mouse so if I tried to label them at the same time with the same green secondary, I couldn’t tell the difference! So ultimately we thought we might be able to do the detection sequentially rather than simultaneously if we fixed the tissue in between.

I’m going to stop the detailed description here but I think you can get the flavor of what I’m doing well enough to see that for a working scientist many experiments are intended to see if you can do what you want with the materials you have. Many preliminary experiments are needed to get to the point of doing the “money” experiment, the one that answers your question or confirms your hypothesis and goes into your published paper.

Wish me luck. I’m going to try a variation of last week’s experiment later today.

We spent time with Kay and John Meisch in Acadia California on our way here. Kay is a Keuka graduate, retired high school chemistry teacher and a member of the Keuka Board of Trustees. We visited the La Brea Tar Pits (Page Museum). Molten asphalt trapped the animals and preserved the bones and teeth of extinct mammals such as saber tooth cats, giant ground sloths, mastodons, mammoths and dire wolves. These Tar Pits are located in the middle of downtown Los Angeles! The museum had an interactive exhibit by which you can experience (by trying to pull on a metal rod) the powerful suck on the asphalt. Animals that experienced the suck directly did not recover from the experience but their bones are preserved for us to see and for scientists to study. These specimens allow us to understand extinct large mammals that evolved after most of the familiar dinosaurs disappeared. Plants and smaller animals are also preserved so the ecosystem can be reconstructed. We saw many reassembled skeletons and a pit from which they were still excavating bones! It was particularly interesting to see mastodon and mammoth skeletons side-by-side and see some of the differences; mostly the teeth (shaped differently) and the tusks (longer in mammoths). Did you know that elephants (and mammoths) replace(d) their teeth up to a total of 6 and when the last pair wears out, they can’t eat anymore? I was so enthralled I forgot to take any pictures. I’ve “stolen” a few from the Page Museum web site but unfortunately they don’t have any skeletons.

The same night we went to see a play at the Pasadena Playhouse. The play was entitled Orson’s Shadow. It was about an interaction between the actors Orson Wells, Laurence Olivier, Vivien Leigh and Joan Plowright. I enjoy “old” movies so I know quite a bit about these four people. Even if you are considerably younger than I you may know Vivien Leigh as Scarlett from Gone with the Wind. Anyway the play was mostly about the different personalities and approaches to acting and directing of Wells and Olivier and about Olivier’s relationships with the two women; his first and second wives. I was familiar with two of the actors from TV shows. The actress who played Vivien Leigh very effectively captured the desperation and hypersexuality that often accompanies bipolar disorder. The actor who played Orson Wells was witty; funny in the way that I think Wells probably was himself. The play has had several revisions and my assessment is that it still could use work; particularly because a younger audience would lack the historical background necessary to care about the characters. I’d give her an A+, him and A- and the playwright a C for effort. Just can’t get away from the tendency to evaluate and score even outside my area of expertise.