At least they are not chewing on each other

I left my rats in the lab overnight so that I could keep tabs on them to make sure that they recovered properly. They are all alive (but not kicking - they don't have a sciatic nerve anymore) but the wounds on three of the rats have dehisced. That means that I am going to have to re-anesthetize them and resuture the wounds. I purposely used a non-absorbable suture material this time but the rats still managed to chew through the stitches. I am going to have to contact the university vet to see what he suggests.

Back to Merlin

Today I am starting another experiment designed to look at Merlin phosphorylation in deenervated schwann cells. It is a relatively easy experiment. I have four rats - two controls and two that will be treated with a PKA (protein kinase A) inhibitor. I will begin by making an incision in the right rear leg and cutting the sciatic nerve. I will then suture the wound up and let the rats recover for one week. Next Thursday, I will go back in, insert an osmotic pump that will release my PKA inhibitor over the cut nerve for 24 hours, and resuture the wound. On Friday, I will remove the pump, and take samples of the nerve proximal (closer to the midline of the body) to the cut and distal to the cut as well as taking the uncut contralateral nerve. Samples will be taken both for microscopy and protein analysis. The purpose to this experiment is to see if Merlin phosphorylation is controlled by PKA when a nerve is cut. We know that when a nerve is cut, the axon distal to the cut dies. This stimulates schwann cell proliferation. Our hypothesis is that PKA, at least in part, is responsible for this stimulation. Previous data have shown that PKA causes Merlin (a tumor supressor gene) to be phosphorylated. When Merlin is phosphorylated, it becomes inactive thereby allowing a cell to re-enter the cell cycle. We want to see we can prevent Merlin phosphorylation in the distal nerve section by treating with a PKA inhibitor. The first time we did the experiment there wasn't any difference between treated and control nerves. I think that was more a protein concentration problem when we loaded the gels but I could be wrong. The second time I did this experiment, the results were inconclusive. Hopefully the third time will be the charm.

I just finished the first round of surgeries and all of the rats are still alive. Hopefully they will be tomorrow also.

Slow week

This has not been the most productive week. I ended up going home early on Monday because my lower back hurt too bad to sit down. Yesterday I set up a maxi prep (a method of havesting DNA by inoculating 100 mL of media with bacteria that contain your gene of interest) but when I came in this morning, the culture media was as pristine as it was yesterday - no growth. I am not sure why the cultures didn't grow except maybe I over treated with ampicillin. I doubled the AMP concentration in an effort to decrease the number of false positives. A friend of mine told me he regularly does that so I thought I would give it a try. Whether that is the problem or not, I don't know.

When I came in yesterday morning, the lab was a mess. Our post-doc had done a protein isolation from a plate of primary schwannoma cells and had left everything out. Apparently she did the experiment on my bench because that was where the majority of the mess was. I am getting sick of having to clean up her messes. I have spoken gently to her about the necessity of cleanliness in the lab but apparently she is not listening. I have worked with quite a few foreign scientists over the past 10 years and most of them have been very good. Several of them, mostly Chinese, have been quite sloppy though. I am not sure if they leave messes because they are used to people like me cleaning up after them or if that is the way they are. This is not just a Chinese thing though. I have found that a good number of physician/scientists are the same way. Maybe they are just used to having nurses around to clean up their messes. I guess I shouldn't pick on my Chinese collegues too much, though. I have known a lot of American scientists who are so messy that I can't stand to even go near their benches. Several weeks ago in lab meeting I brought this problem up by telling everybody that I really didn't care what their own work space looks like but I do care what our shared work spaces look like. It is simply common courtesy to clean up after yourself - didn't their mothers (and fathers) teach them that? How can you do good science in a pig sty?

Grants

We had a bit of a set back yesterday. We turned in a grant application to the National Institutes of Health in Feb. The NIH is made up of many smaller sections and when you turn in your grant, it is assigned to one of the sections - usually the one deemed most appropriate based on your grant. Our grant kind of straddled three of the sections - cancer, audiology, and neuroscience. We asked that it be assigned to Audiology but someone got the great idea to assign it to cancer. That means that we are competing for a fist full of dollars with people studying breast cancer, lung cancer, prostate cancer, and the like. Our cancer doesn't affect or kill anywhere close to the number of people as the ones I just mentioned. As such, the chances of our grant getting funded with Pres. Bush's extremely tight grip on the NIH are non-existant. Truthfully speaking, those cancers do deserve the bulk of the funding from the Cancer section of the NIH. That is why we applied to a different section. In the last 15 years, the Cancer section has not funded one grant on Vestibular Schwannomas. After hearing that we were being assigned to the Cancer section, we asked that the grant be reassigned but to no avail. We are considering reapplying to the Veteran's Administration for funding. I guess we'll have to see what happens. NIH grants have been very hard to get the last few years. Even well established researchers are getting their grants tossed.

Eventhough we have two currently funded grants, we are always writting the next one. This is a never ending process. I am grateful the responsibility for funding a lab does not fall on my shoulders. Research is very expensive. Even basic consumables are expensive. The only thing that comes cheap is the labor. Although I like to complain about my pay, I really am lucky to have this job. I really do love what I do. I guess there really isn't much to complain about.

Well, my timer goes off in 12 minutes so I need to wrap things up and get back to the next step of my Western blot.

Slower than a snail

Research goes at a snail's pace. For every "discovery", there seems to be at least 10 setbacks. I guess if it worked every time we could just call it search instead of research. I have more Western blots to do this week. Hopefully I can get these last few blots wrapped up so that we can finally get this paper submitted. We are going to try to get it published in the Journal of Biological Chemistry (JBC). I have my doubts that we will be successful getting it into JBC but it is good research so hopefully we will have a chance. Journals are rated first tier, second tier, or third tier (not offically, though) depending on the influence of the journal. Journals like Science and Nature are considered tier 1+, JBC, the American Journal of Physiology, Cell, and the like are considered tier 1 journals and on down from there. I don't like a lot of what comes out of Science and Nature because these are the breaking reseach stories and due to space limitations, I can never get all the information I want. Also, in the excitement to publish ground breaking research before someone else does, the experiment(s) is not repeated as often as it should be. This leads to retractions. There are probably more retractions in Science than any other journal. Having said that, retractions are still few and far in between.

I won't get much done today. I attended a funeral this morning for a friend that died last week. I am glad I got to go but I just don't have the time I need to run another gel. Hopefully I will get the rest of the blots run this week so that I can move on. I have some cloning that I need to do. It has been hanging over my head for months. When it works, its great but when it doesn't it is very frustrating. One simple base pair misalignment and the whole thing goes down the drain.

More fun with Westerns

I have been doing a lot of Western blots lately and thought that I would show a picture of one so that you at least have an idea of what I am talking about. The blot I am showing you is actually three blots - one looking at phopho-AKT, another at phospho-JNK, and the last (far right) at phospho-ERK. There are two lanes in each blot, one is of tumor sample, and the other of control tissue. This has been my life the last couple of weeks. The tumor samples are showing darker bands but that is probably because there is more protein in the tumor lanes than in the control lanes rather than an actual upregulation of enzyme activities.

I can't help but laugh sometimes

More protein problems. Actually the proteins are doing what they are supposed to, we are the ones with the problem. Our post-doc did a Western blot of her protein purification process and found a protein that was enriched in a couple of the elution fractions just like she saw on her silver stained gel. That is good except the protein band appeared to be about twice as large as it should have been. We think that that is probably because this protein likes to self-dimerize. However, we don't want the protein to be dimerized. I had her do a couple of things that would solve the problem and after running the blot again, she excitedly showed me the film (Western blot results are recorded either digitally or by film) that showed protein bands much lower on the blot. After taking one look, I saw from the shape of the bands that she had the film upside down. She assured me that she didn't, showing me where she had creased the film to ensure proper orientation just like I had taught her. Sure enough, her mark was in the correct place but she had placed the blot upside down in the film cassette. I managed to only grin a little bit while explaining the problem. I need to sit down with her and help her design the next couple of experiments to improve our yield and purity as well as our dimerization problem. These next few experiments are going to be a bit more involved but should yield good results. Of course now that I have said that, I have cursed the experiment. We'll see.

Results are always good

I finished another Western blot, well, actually 6 of them. The results were encouraging I think. I am not sure what I expected. I ran two gels with control tissue (2 different Greater Auricular nerves - they are sensory nerves that transmit touch from the ear). I transfered the proteins to nitrocellulose membranes and incubated them with antibodies raised against Erk, AKT, and JNK, as well as the phophorylated, or activated, versions of those three signalling proteins. Both Erk (pronounced irk) and AKT are generally considered to be enzymes involved in proliferative signalling pathways while JNK (pronounced junk) is considered to be a player in cell death pathways. The non-activated versions showed equal amounts of protein between both samples - thankfully - but only AKT was activated in both samples. Now I need to go back a do the same experiment with my vestibular schwannoma samples so that I can compare the two. This will give us the beginning of an idea as to what signalling pathways are activated in this tumor. We have so much work to do and of necessity it crawls at a snail's pace.