tag:blogger.com,1999:blog-64352396246089813662023-11-16T02:48:18.219-08:00Biology, Cell Biology, Biochemistry, and ScienceA study guide to all things collegiately biological (i.e. all your core courses for a science major).Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.comBlogger44125tag:blogger.com,1999:blog-6435239624608981366.post-4141906359144992172013-05-01T09:48:00.001-07:002013-05-01T09:48:05.835-07:00Correction to q22 Stem Cells Final Review QsThanks Sydney for pointing out that Dr Brown did include mention of evidence of cancer stem cells in breast cancer -> on one of his slides, there is described an experiment of transplanting cells taken from existing breast cancer into a mammary pad and observing that it grows into another tumor.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-30735969249366730952013-04-28T20:37:00.005-07:002013-04-28T20:37:44.724-07:00Stem Cells Final Review QFirst off, I've started putting down the lectures that you'd look at to figure out the answers. As I progress on my own last minute studying, I'll try and add bulletpoints of key things you probably could mention in your answers. Also if you have suggestions I'm happy to put them up too (comment below!)<br />
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https://docs.google.com/document/d/1IyCV-7EdsXEt_QiSA2cCjiJC_ckiD8yRuJ8W1t3KR38/edit?usp=sharing<br />
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Good luck!Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-71948678037103892612013-04-27T17:01:00.003-07:002013-04-27T17:01:43.391-07:00More Research Paper Sumups!I will be focusing on the study guide from here on out. If I am done that early and have extra time, I will continue the research paper sum-ups. Until then, good luck with studying!<br />
<h3>
Savitt J, Singh D, Zhang C, Chen L, Folmer J, Shokat K, and W Wright. "The In Vivo Response of Stem and Other Undifferentiated Spermatogonia to the Reversible Inhibition of GDNF Signaling in the Adult." Stem Cells 4, 2012: 732-740.</h3>
Intro:<br /><br />There's evidence that GDNF is important for regulating population size of spermatogonial stem cells.<br /><br />What is the evidence? For a long term culture, you need GDNF to maintain and expand stem spermatogonia (how do you know they are stem spermatogonia? by putting them in a testis w/o germ cells and restoring spermatogenesis).<br /><br />But wait, is GDNF just a survival factor or does it actually promote the self-renewing kind of replication for stem cells? Let's see. Mouse stem spermatogonia were deprived of GDNF for 3 or 6 days, but the population still grew as if it had GDNF. One would infer from this that GDNF does not promote self-renewal over differentiation. So that's the in vitro study.<br /><br />What about in vivo? We are kinda limited to knocking out or messing with GDNF expression from time of birth or earlier, but not in adult tissue, nor can we try to restore it after knocking it out to see what happens. Overexpression of GDNF -> lots of undifferentiated spermatogonia that just died by apoptosis and led to infertile adult. Knockout of GDNF -> lost all germ cells in a week and the few left did not replicate.<br /><br />Lots of questions, but how to answer them? We made a new system where we could inhibit GDNF and also reverse the inhibition. Let's look at the signaling pathway. GDNF binds Gfr-alpha1 which activates Ret and phosphorylates Ret (also part of the same receptor). Our transgenic mice had 1 mutation in Ret but it doesn't affect Ret activity. BUT it increases affinity for a competitive inhibitor NA-PP1. So by treating mice with NA-PP1, it competes with ATP and prevents the binding of GDNF from phosphorylating RET. When you take away NA-PP1, things go back to normal.<br /><br />hypothesis: GDNF is essential for maintaining spermatogonial stem cell pool and also stimulates self-renewing replication of the stem cells. prediction if hypothesis is true: stem spermatogonia will be lost if GDNF signaling is inhibited for duration of 1 cell cycle (2 days).<br /><br />Results:<br /><br />the mutation in Ret works: baby mice with underdeveloped kidneys die with the mutation (proves mutation works), but it doesn't affect adults (proves it's usable for studying adult tissue)<br /><br />ok, so treatment with NA-PP1 for 5, 11, 20 days and looking at Gfr-alpha1 positive cells and Zbtb16+ cells (these are regular spermatogonia, not stem spermatogonia). the Gfra1+ cells are less and less by 5 and 11 days and basically gone by 20. The Zbtb16+ aren't changed at 5 but are reduced by 11 and gone by 20 as well. This doesn't happen in mice w/o the Ret mutation. similar results seen with mRNA levels of Ret, Gfra1, and Zbtb16.<br /><br />Still, after 20 days, those transcripts are still detectable (just reduced). Did we lose functional stem cells or did we just reduce them to numbers so low they're hardly detectable? Let's do 30 days treatment and collect the tissue either then or 35 days later. Histology sections show Ret-mutated mice lost ALL their spermatogonial stem cells from 30 days of treatment with NA-PP1. Further testing showed this happened over a course of several days (not all at once)<br /><br />After the 11 day treatment though, some still remain! 35 days after the 11 day NA-PP1 trtmt, testes were back to normal. What? We saw 3% of tubules active in spermatogenesis and there were dense clusters of Gfra1+ cells. We also saw that even with a short 2 day NA-PP1 trtmt, some stem spermatogonia are gone.<br /><br />Discussion:<br /><br />This is the first use of a chemical-genetic approach to study adult stem cell regulation via growth factor. This method has 3 advantages:<br />1) you start with totally normal pool of stem cells (existing in normal organism, not in weird imitation culture, etc)<br />2) you get normal in vivo response to whatever you are doing<br />3) you can reverse what you just did and go back to normal conditions<br /><br />We showed GDNF is required for maintaining stem cells in adult testis. Also we saw stem cell loss with absence of GDNF, which is opposite to the stem cell growth mentioned earlier in that in vitro study. But some survive even past 11 days, so there are definitely other factors involved.<br /><br />This may be due to a) varying normal concentrations of GDNF for various stages of the tubules, b) the cells normally do not replicate at the same time all together, c) it has been hypothesized that there are quiescent ones also who enter the cell cycle only when tissue damage has occurred.<br /><br />When NA-PP1 treatment ceases, cells migrate and refill empty areas, and new refilled niches will start producing spermatogonia again with time.<br /><br />
<h3>
Stanley E, Lin C, Jin S, Liu J, Sottas C M, Ge R, Zirkin B, and H Chen. "Identification, Proliferation, and Differentiation of Adult Leydig Stem Cells." Endocrinology 153, 2012: 5002-5010</h3>
When testes get depleted of their Leydig cells (testes interstitial compartment) with treatment of EDS, they come right back! There must be cells that make Leydig cells then, but very little is known about them.<br /><br />A previous study isolated cells from new baby rat testes that could divide w/o differentiating or divide and produce testosterone (normal Leydig cell function). And when they were put into normal testis, the cells also could differentiate -> evidence that these baby rat cells were Leydig stem cells.<br /><br />This study looks for stem Leydig cells in the adult testis. Since it is possible to separate seminiferous tubules and interstitial compartment of a testis, it made it feasible to go looking for these stem cells and their physical niche.<br /><br />Results:<br /><br />Testicular cells were isolated from Leydig-depleted testis and grown in culture. Without LH (luteinizing hormone), cells kept growing and growing for about 1.5 yr (325 population doublings). They didn't produce testosterone. When cultured with LH, they produced testosterone.<br /><br />Seminiferous tubules and interstitial compartment were separated from Leydig-depleted testis and cultured separately. Cells appeared on surface of cultured tubules but not cultured interstitial compartment. These cells stained for 3B-HSD (enzyme in pathway of making testosterone) and the tubule culture, when treated with LH, started producing testosterone (neither tubule culture w/o LH nor interstitial culture w/ or w/o LH made testosterone). Therefore, the Leydig precursor cells came from the tubules.<br /><br />Are they stem cells? These cells were selectively labeled with EdU, and then given EDS, which stripped the 3B-HSD plus label cells. Some time later, they came back. If you measured testosterone, its presence and absence correlated with the presence/absence of the 3B-HSD cells.<br /><br />Discussion:<br /><br />Had been known previously that Leydig cells came back after depletion and it was the point of this study to see if there were stem cells ( as opposed to quiescent progenitor cells ) and where they are and how they are regulated.<br /><br />If they were stem cells, we should have been able to isolate them and have them grow for a long time without trying to repress any Leydig cell markers and be able to differentiate into T-producing cells. We accomplished this.<br /><br />There have not been any Leydig stem cell markers identified, so we had to go a different way about locating them. Our results with the separation of tissue in culture experiment demonstrated they were the cells growing on the surface of the tubules.<br /><br />If they were stem cells, they should be able to come back again if they suffered another depletion by EDS. The EDS in the tissue culture experiment showed that. Also we had an unpublished experiment where we treated the tubules with collagenase and dispase to cut anything attached to surface of tubules off, and any stem-activity was lost (couldn't make cells that produce T).<br /><br />In terms of regulation, more research is needed. But considering that they could grow on tubule without the interstitial compartment present suggests the blood vessels associated with interstitial compartment are definitely not important, but they might still have a function in vivo.<br /><br />Also the tubule culture method is a great way to keep answering questions about niche components in regulating adult stem cells.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-55983928751506893802013-04-27T12:29:00.003-07:002013-04-27T12:29:27.466-07:00Two more paper sumups: Germline Stem Cells<h3>
Eun S H, Gan Q, and X Chen. "Epigenetic Regulation of Germ Cell Differentiation." COCeBi 22, 2010: 1-7.</h3>
<br />Epigenetics modify chromatin w/o affecting DNA sequence. There are proteins who "write" on the modifications, "read" and act on the modifications, or "erase" the modifications.<br /><br />Germ cells are "immortal" b/c they produce the next generation. Epigenetics in germ cells might be involved in<br />1) meiosis and terminal differentiation<br />2) keeping the genome information good<br />3) erasing bad information<br /><br />GSCs (germline stem cells) initiate gametogenesis. Their chromatin structure maintains self-renewing and blocks differentiation. Evidence: ISWI "Imitation Switch" is a chromatin remodeling factor. it is essential for preventing differentiation (in mutants for iswi, the bag gene is expressed and induces differentiation). One example is the NURF complex that contains the ISWI factor in male drosophila.<br /><br />histone modifications also functions in maintaining stem-cellness. Evidence: scny gene removes ubiquitin from histone H2B. scny mutant leads to super acetylated H3 and super ubiquitinated U2B => open or loose chromatin -> active transcription of differentiation genes.<br /><br />also, this chromatin landscape isn't permanently blocked from differentiation but described as "poised" to start differentiation upon stimuli. in mammalian: have both activating H3K4methyl3 and repressive H3K27methyl3 going on and a paused RNA Pol II. it prevents DNA methylation, which is a more stable block and would be difficult to remove for differentiation to happen at a snap. in drosophila: not bivalent (i.e. not an active and a repressive histone modification at the same time) and not associated with paused RNA Pol II, but probably b/c drosophila doesn't have methylase anyway.<br /><br />only germ cells do meiosis. with meiosis, chromatin regulators and opposite-fxn histone modifying enzymes are downregulated. at same time, celltype specific factors are unregulated (i.e. the tTAFs testis-specific TBP-associated facotrs in drosophila spermatocytes)<br /><br />same/similar-fxn histone modifying enzymes work together though, maybe separated by time (i.e. in drosophila ovary, one isoform is in early germ cells and GSCs while the similar isoform is needed in later stage germ cells). but in mice, meiosis was found to have both functional enzymes that are opposite fxn.<br /><br />also during mammalian gametogenesis is when imprinting (DNA methylation) happens. This might happen via a sort of weird transcription process that goes over modified genes in order to "read" and match it to the new DNA strands. This happens while oocytes are arrested in meiosis but we're not sure about spermatocytes. More research needs to be done.<br /><br />in spermiogenesis in drosophila and mammals, histones are kicked out and replace with Tnps and Prms to super condense the DNA in super nuclei. This process is also regulated by epigenetics. Some examples include:<br />1) H4 S1A substitution/mutation messes up phosphorylation of H4S1 and results in failed sporulation in yeast<br />2) demethylation of H3K9me2/1 is necessary to increase expression of the Tnps and Prms in mice.<br />3) (see more in the paragraph)<br /><br />in human sperm, about 4% of the genome still has regular histone nucleosomes, no Tnps or Prms or anything. Specially, the Hox genes and imprinted genes that are necessary for early embryonic development. but things like Nanog that are necessary for ESC pluripotency are off.<br /><br />after fertilization, Prms get kicked out and given maternal histones. some chromatin regulating proteins are involved in helping out. the cells that will be the germ cells for this zygote's babies are the PGCs (primordial germ cells). They get a genome-wide erasure of DNA methylation so as to get rid of any epimutations that happened between their ancestor's PGCs and this fertilization.<br /><br />Proper histone modification control takes care of differentiation genes and fertility, but they also have been observed to ensure proper lifespan.<br /><br />During the journey of germ cells, they need to maintain a correct epigenome, which is ensured by dynamic control, erasure, and re-establishment of the epigenome. More needs to be done in the field, except it is hard to get a good amount of these GCs and PGCs (ethics!) so if we can has new technology that don't need as many cells, we will be good to go. Also this will be useful for diseases due to germ cell differentiation fail (infertility, germ cell tumors, etc) but also regenerative medicine.<br /><br />
<h3>
Tran V, Lim C, Xie J, and X Chen. "Asymmetric Division of Drosophila Male Germline Stem Cell Shows Asymmetric Histone Distribution." Science 338: 2012: 679-682.</h3>
<br />question: stem cell activity is regulated by epigenetic, but do stem cells retain their epigenetic info?<br /><br />male drosophila GSCs have been well characterizes and are easily identified. they divide asymmetrically and can be examined at the single cell level.<br /><br />histones are major carriers of the epigenome. we created this switchable dual-color method to label and identify what is a new histone versus and old histone. can be controlled spatially or temporally (by heat shock!). labeled histones can get switched from green to red.<br /><br />how? heat shock causes the recombinant gene to irreversibly shut down GFP-labeled old histone expression and initiate expression of new mKO-labeled histones. if old histones are distributed btw daughter cells equally, over time any green color will be replaced by red color. if old histones are kept in the GSC and new histones are kept in the daughter fated cell, then green will be specifically retained.<br /><br />before heat shock: testes with transgenes showed green nucleus but almost no red. after heat shock, red started appearing. of all GSCs, about 70% of them will be in G2 phase, 21% in s phase, and <2% in mitosis. entire cycle length is 16 hrs. plus the two daughters will be connected after incomplete cytokinesis and are called "spectrosomes." examine testes 16-20 hrs after heat shock, specifically the spectrosomes.<br /><br />results: green preferentially held in daughter GSC, while red was preferentially delivered to fated daughter cell. in contrast, for symmetrically dividing GSCs, both daughters had equal red and green. also in contrast, this was observed with canonical H3 transgene, but not with variant H3.3 transgene. also looking at during mitosis, anaphase pulled green chromatids toward future daughter GSC while the spindle pulled red chromatids towards future fated daughter cell.<br /><br />change it up: ectopic activation of JAK-STAT signaling pathway. previously shown that this causes overpopulation of GSCs. they did it and found that the asymmetric green-red distribution didn't happen. so equal distribution of old and new histones is also associated with this forced symmetrical divisions.<br /><br />proposed mechanism: old and new histones are incorporated into separate chromatids during S, and are preferentially segregated at the spindle.<br /><br />this may go towards understanding how epigenetic is maintained in SCs and reset in sibling cells that will differentiate.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-88012138574050733352013-04-27T10:23:00.002-07:002013-04-27T10:23:39.130-07:00Prepping for Final Exam of Stem Cells BiologyOk, so the doc has been updated with ALL the lectures of this semester now, hopefully typo free. Again, comment or email me if you have questions or something looks funny.<br />
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https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharing<br />
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Sumups of the other research papers (16 more to do, homg) and a study guide for the exam coming either later today or tomorrow. Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-50730442691061224762013-04-04T19:45:00.001-07:002013-04-04T19:45:35.390-07:00Audio of Stem Cell Lecture 7, Lecture Transcript updatesHere's the audio for lecture 7 with Dean Gregory Ball. I will be discontinuing the audio recordings b/c I simply don't have the equipment for good recordings (I did Ball b/c he has such a booming voice anyway).<br />
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https://dl.dropbox.com/u/22080433/balllecture.mp3 <br />
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But, also I have updated the lecture transcript with Lecture 7 and 8, Dean Ball and Dr. Alan Meeker.<br />
https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharingAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-18300169104125557652013-03-17T16:43:00.000-07:002013-03-17T16:43:46.541-07:00Audio of Stem Cell Lecture 6 and Reminder Link of LectureTranscriptsWell, here's audio lecture of Number 6, Dr. Daniella Drummond-Barbosa (JHSPH) on Stem Cells in Drosophila Ovary <br />
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https://dl.dropbox.com/u/22080433/march13lecture.mp3<br />
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and the link again for the lecture transcripts: <br />
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https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharingAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-2993247075003689772013-02-27T13:52:00.003-08:002013-02-27T13:52:57.241-08:00Audio Recording of Stem Cell Lecture (attempt 2)So here I have attempted to simplify by just taking an audio recording of the lecture by Dr. Barry Zirkin (this time on Leydig stem cells). I have to forewarn you that my computer's Audacity program also succinctly captured the sound of my typing (as I transcribe the lecture as well). Apologies if that annoys you :(. Still, it captures the lecture all the same.<br />
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https://dl.dropbox.com/u/22080433/StemCell2013Lecture5.mp3<br />
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Also I still update the transcribed lectures on the google doc (see link some posts earlier) and in two weeks, we have our first exam, so in one week, look out for some study guides! :DAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-20024415917490833782013-02-27T11:59:00.001-08:002013-02-27T11:59:30.070-08:00Lecture 2 Paper Sumup: induced Pluripotent Stem CellsTakahashi K and Yamanaka S. "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors." <i>Cell</i> <b>126</b>, 2006: 663-676.<br />
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Introduction:<br />
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ES cells are pluripotent: they come from mammalian embryo and can make cells from all 3 germ layers.<br />
- super useful for treating diseases<br />
- but ethically controversial (can we use human embryos for this purpose?)<br />
- and physically difficult if implanted tissue is rejected by host<br />
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possible solution? get pluripotent cells from host themselves!<br />
- take normal body cell (somatic cell) DNA and stick it in an oocyte or fuse the cell with an ES cell<br />
- the other cell contents of ES cells/oocyte have been shown to contain some factors that make somatic cells pluripotent!<br />
- we know some factors that maintain pluripotency, but maybe these factors also INDUCE pluripotency<br />
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what do we know about factors that maintain pluripotency:<br />
- Oct3/4, Nanog handle maintenance<br />
- Stat3, E-Ras, c-myc, Klf4, Beta-catenin are highly expressed in tumors--they also help maintain long term ES cell phenotype and proliferation<br />
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Results:<br />
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We tried out 24 genes that could have been the factors that induce pluripotency.<br />
- experiment: if gene X induces pluripotency, cell will be resistant to G418 (a molecule that inhibits protein synthesis).<br />
- ergo, if we see cell resistant to G418, then the gene that we unregulated in the cell is a factor that induces pluripotency.<br />
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step 1: in mouse, knock out the gene Fbx15. This gene is super important for maintaining pluripotency in mouse development.<br />
- ES cell with knocked out Fbx15 resist G418<br />
- somatic cells with knocked out Fbx15 cannot resist G418<br />
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step 2: one by one, insert each of the 24 candidate genes into these knockout embryonic mice cells<br />
- no resistance observed<br />
- ergo, these genes cannot induce pluripotency by themselves<br />
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step 3: upregulate all 24 genes in these knockout cells<br />
- get lots of resistant colonies!<br />
- some of these look very similar to ES cells (morphology, proliferation traits, gene expression markers, etc)<br />
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step 4: upregulate all but 1 gene in these knockout cells<br />
- found 10 genes that, once you did NOT upregulate them in the cells, you did not get resistant colonies<br />
- ergo, these are super important factors that you can't NOT put in in order to induce pluripotency<br />
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step 5: upregulate all 10 special genes in knockout cells<br />
- get lots of resistant colonies!<br />
- many of these look similar to ES cells<br />
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step 6: upregulate all except 1 of these 10 special genes in knockout cells<br />
- not including either Oct3/4 or Klf4 resulted in no resistant colonies<br />
- not including Sox2 resulted in very very few resistant colonies<br />
- not including c-myc resulted in weird looking resistant colonies<br />
- not including any of the others produced all resistant colonies, so the others were not as important as the above 4.<br />
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step 7: upregulate only those 4 super special genes in knockout cells<br />
- get same result as step 5<br />
- culture and confirm that these are iPSC (induced pluripotent stem cells)<br />
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step 8: upregulate pairs and triplets of these 4 super special genes in knockout cells<br />
- no two of them could form any resistant colonies<br />
- 2 triplets produced a few colonies but they did not survive further culturing<br />
- 2 other triplets produced more colonies but looked weird (different from ES or previously determined iPSC)<br />
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step 9: do gene expression analysis of iPSCs induced with the various combos<br />
- the 4 combo and the 10 combo cells, both are similar to ES cell expression profiles but not exactly the same<br />
- the 3 combo cells were very very different<br />
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step 10: try to make teratomas with these various combo-formed iPSCs<br />
- there was inconsistent data: some of the 10 combos and the 4 combos made teratomas with cell types of all 3 layers, but some of the same combos could only form 2 or 1 of the germ layers<br />
- so conclude the majority of the 10-combo and 4-combo cells are pluripotent, but not all<br />
- tumors from 3-combo cells did not differentiate = not pluripotent!<br />
- similar results when trying to form embryoid bodies in culture as opposed to forming teratomas in vivo.<br />
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step 11: introduce the 4 combo gene into mouse tail fibroblasts (somatic cells) and then inject these cells into blastocyst<br />
- were able to observe that these injected cells helped form some of the germ layers and baby mice were actually born from these blastocysts that received injections!<br />
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step 12: compare gene expression levels of these 4 factors with protein expression levels btw iPSC and ES<br />
- saw that while some of these genes were higher or lower in iPSC cells than in normal ES cells, the protein levels (Western blot!) were about the same!<br />
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step 13: try to grow iPSC without them differentiating in culture<br />
- they always differentiated unless they were provided "feeder cells" in the same culture<br />
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Discussion:<br />
- Oct3/4, Sox2, Nanog are essential for maintaining pluripotency<br />
- Oct 3/4 and Sox 2 are essential for MAKING iPSCs<br />
- Nanog is not important for that<br />
- c-Myc Klf4 are also essential<br />
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- c-Myc upregulates genes for proliferation and transformation<br />
- it affects some histone modifying enzymes (histone acetyltransferase, for example)<br />
- there are a LOT (upt to 25000) of sites for c-Myc binding in mammal genome<br />
- this is way more than what we'd guess for Oct3/4 or Sox2 binding sites<br />
- it could be that c-Myc causes global histone acetylation, causing a lot of the genome to open up, so that Oct3/4 and Sox2 can find all their target binding sites<br />
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- what about Klf4? represses p53<br />
- okay, what does p53 do? It suppresses Nanog during differentiation<br />
- so if you repress p53, you enable Nanog, which should normally NOT be active for differentiation.<br />
- this might contribute to making the iPSC or at least ES-like cell phenotype<br />
- Klf4 activates p21 which suppresses proliferation, and c-Myc suppresses p21. This opposites relationship of c-Myc and Klf4 might be important (in other words, we're just guessing)<br />
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- one important question: which cells of the tissue given these four factors are becoming iPSCs?<br />
- only a small portion of cells treated with the 4 factors become iPSCs<br />
- maybe it's the progenitor/stem cells that already exist in tissue that are kinda multipotent but not pluripotent that transform into pluripotent cells<br />
- the frequency doesn't change when we try this out with bone marrow, which should have a high percentage of progenitor/stem cells to begin with to change<br />
- so it can't be those cells..<br />
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- maybe getting the right expression level of each factor in the cells is important<br />
- experimental evidence: just a 50% increase or decrease in Oct3/4 proteins in an ES cell causes it to differentiate and lose pluripotency<br />
- we know our iPSC clones overexpress RNA levels but their protein levels of the 4 factors are <i>just right</i><br />
- but these cells must be able to regulate that, b/c high high levels are necessary to become ES-cells but in order to <i>stay</i> ES-like, too much of the 4 factors is badddd<br />
<i>- </i>they might need some chromosomal alterations too to stay ES-like<br />
- this may be spontaneous or induced by some of the 4 factors<br />
- where the retrovirus brings in the transgenes to overexpress the 4 factors also matters: could have impacted the expression of any native genes depending on how the transgene got shoved into the genome<br />
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- another question: are these 4 factors also important when we're trying to reprogram somatic cells by fusion with ES cells or plucking out nuclei and putting them in oocytes?<br />
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- the precise roles of Klf4 and c-Myc are also confusing and vague. they aren't essential for mouse development before the egg implants. c-Myc isn't detectable in oocytes at all. Hmm??<br />
- well, related proteins, L-myc and Klf17 and Klf7 do exist. maybe Klf4 and C-myc's real properties are being supplanted by these relatives in wildtype development<br />
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- some other questions that this paper brings up...<br />
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- still unsure if these 4 factors can make pluripotent cells out of <i>human</i> somatic cells.<br />
- testing/experimental process is going to require super specific culture environments<br />
- but this is all really cool in the search for the tools to control pluripotency, and one day we might be able to make pluripotent cells from a patient's somatic cells.<br />
<br />Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-28986753713458874802013-02-24T11:09:00.001-08:002013-02-24T11:09:29.513-08:00Stem Cells: Lecture 4 VideoHere is my first attempt at recording. I did it less for the purpose of showing slides (because we get that provided at our school) and more for the purpose of audio learners, who don't do well just reading the transcripts of the lectures. I might just try an audio recording instead of video and audio next time.<br />
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This is the lecture by Dr. William Wright on the subject of stem spermatogonia. <br />
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https://www.youtube.com/watch?v=br7STDDbx24Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-52931114963743280602013-02-21T08:23:00.000-08:002013-02-24T11:10:06.454-08:00Papers, videos, and transcriptsHello my Stem Cell Biology people!<br />
<br />
First of all, remember to keep checking the google doc of the lecture transcripts. I always update it ASAP and I've been trying to correct my spelling mistakes and add in a few more explanatory words in case I'm not sure the lecturer was clear on something.<br />
<br />
Also I'm experimenting with recording the lectures, b/c for some, they are better audio learners. Bear with me as I struggle with technology.<br />
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Finally, we have one or two papers assigned for every lecture, so I'll post a sum-up of the papers. I still recommend you read the papers and then read the sum-up, because 1) you need practice if you ever want to be able to digest the condensed jargon of science writing and 2) the more you read the material over and over again the more it sticks in your head.<br />
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Here it is from Lecture 1 (Please excuse my casual language. It is my opinion that to make science available to the general, you must speak "layman"...seriously though. I am not trying to condescend to anyone, I just want to make sure people can understand, because that's the foundation to building up knowledge later on.)<br />
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Also remember that every paper wants to tell you something, but it is always up to you if you choose to follow or take it for its word. Always ask questions (is this right? did they do this correctly? did they consider this? etc etc) Whatever I write below is not an expression of something I believe in, but just a paraphrase of what I interpreted the paper was trying to say. I'm always open to discussion! (hence, comment box)<br />
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<span style="font-size: large;"><b>Spradling, Allan C. "The living-tissue microscope: the importance of studying stem cells in their natural, undisturbed microenvironment." J Pathol 2011; 225: 161-162.</b></span><br />
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<b>We need tools to look at cells in their daily lives without disturbing them.</b><br />
Why? Because we would find out all about normal cell lives, diseased cell lives, new cell to cell and cell to gene and gene to gene pathways that we can't see from frozen or dead cells.<br />
<b><br />We have the tools to do this:</b><br />
Small multicell structures with labels can be seen live, for a long period of time (1-3 hr) and at the level of a single cell.<br />
approach referenced from Gaisa et al (see citation 4 in this paper) where they used mitochondrial DNA mutations to trace where each cell came from (lineage analysis)<br />
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<b>This is super important for stem cell research.</b><br />
Why? they are rare and hard to find and hard to identify and basically impossible to label with gene expression tag<br />
But you can do it with lineage analysis!<br />
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<b>How did they do it back in the day without being able to creep on cells in vivo?</b><br />
well, they took tissue from active stem cell tissues (like bone marrow or testis), and put them in a host with little to no stem cell activity, and then voila, cells were being made there.<br />
researchers figured out that there needed to be an area "stem cell niche" to for stem cells to be enriched and function<br />
BUT they couldn't tell if all the cells they transplanted were actually stem cells or just some of them were…<br />
some later studies in flies and mice showed that even daughter cells from stem cells that had begun differentiating (progenitor cells we call them now) could reverse and go back to being stem cell if they found a niche-->hmm, it was now supposed that tissues often contain lots of potential stem cells as well as stem cells.<br />
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<b>But we still can't pick out and identify specific actual stem cells until we can look at them in vivo without disturbance. We still don't know where they are in most mammalian tissues.</b><br />
But we can fix that! We need everyone to do lineage tracing in all of the organs!<br />
<br />
<b>it used to be that people searched for stem cells on guesses and unfounded assumptions:</b><br />
<u>myth</u>: most stem cells are quiet and reproduce only sporadically.<br />
<u> assumption</u>: it must be cells that are still labeled with BrdU (thymidine analog, gets into DNA) after a long time--because they are quiet--must be stem cells.<br />
<u>truth</u>**: at least six types of stem cells have been shown to divide continuously<br />
<br />
<u>myth</u>: stem cells spawn huge numbers of daughter cells and these may even be extremely diverse cells (different cell types) once you stick it into tissue culture<br />
<u>assumption</u>: if we stick cells into tissue and they don't suddenly reproduce a crap ton of cells, they must not be stem cells…<br />
<u>truth</u>: most stem cells are maintained as stem cells only by their niche. if they leave or are pushed out, they stop being stem cells and differentiate.<br />
<u>example</u>: mouse intestinal stem cells need specific cytokines and actual niche cells and other stuff in order to successfully propagate.<br />
<br />
(**Keep in mind that I only say "truth" to mean that there is evidence to the contrary, not that this is the as-laid-down-by-the-laws-of-nature-factual-truth. Remember always to have a healthy dose of skepticism in science.)<br />
<br />
we only figured all that out after the cells were identified by lineage analysis and we could characterize their niche and signals in the niche<br />
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unless we have an exact replica of the niche, stem cell in tissue culture will look nothing like stem cells in vivo.<br />
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<b>This is applicable to fields outside stem cell research.</b><br />
embryo cells have constant signaling and interaction with their environment during development<br />
cells in culture also have change in genes/epigenetics, even if their overall karyotype is still the same.<br />
this is all likely due to the fact that they are stressed in an environment not like what they should be in (different signals, treatments, etc)<br />
this is why tissue culture instead of just cell culture is better for multicellular biology<br />
<br />
<b>lessons learned from stem cell research:</b><br />
if we still want to use cell culture but we want to look at things that don't happen just by themselves, we need new tools<br />
we have to look at the events in vivo or in tissue culture<br />
when we figure stuff out about the cells of interest and their environment, then we can create a replica cell culture<br />
then we gotta double-check our model to make sure cells in vitro are behaving just as we had already observed in vivo<br />
<br />
<b>you have to follow these steps if you want accurate replication of the crazy complicated system of biology that every cell activity depends on.</b><br />
good thing new developments in live imaging and lineage analysis are going to make this easier<br />
just remember that you don't want to destroy the very biological events/systems that you wanted to study in the first place.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-30678463777434053812013-02-17T06:08:00.001-08:002013-02-17T06:08:51.056-08:00Cell Biology Study GuidesSo I realized I must have shared my Cell Bio exam guides elsewhere or in person, b/c it's not on this blog!<br />
<br />
So here they are! Cell Bio exam 1 guide and exam 2 guide. There's already a post for exam 3 somewhere on this blog.<br />
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exam 1: https://docs.google.com/file/d/0B99-sSwVe231OWM2YjA3OGItOTU3Ni00MGFiLWEzYzEtMzViNTczMTljYmY5/edit?usp=sharing&authkey=CLKk8NQB<br />
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exam 2: https://docs.google.com/file/d/0B99-sSwVe231NTI4Mzk1MWMtMGQwYi00M2JlLWFkM2YtMGRkYTM4NDNhYTFl/edit?usp=sharing&authkey=COCampkH<br />
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Again, let me know if the links don't work! (via comment or something)Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com2tag:blogger.com,1999:blog-6435239624608981366.post-11976679768583181332013-02-10T21:35:00.001-08:002013-02-10T21:35:55.523-08:00Stem CellsFor the purpose of the class I'm taking, I'll be making three kinds of posts:<br />
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1) script -> the class at our university does not have podcasting, so I will be transcribing a misspelling-prone version of near-verbatim of the lectures. Please find it here: https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharing<br />
I have little time to spellcheck or grammar check, so please comment if you find something confusing that I can clarify! <br />
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2) study guide -> this will be prepared prior to every exam. hopefully it helps!<br />
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3) list of papers -> a significant portion of this class involves reading primary documentation, so I will be listing citations of the papers we read (and that will contain info that I will probably include in my study guides).<br />
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Ready for stem cells!Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-89239817547928766992013-02-10T17:17:00.001-08:002013-02-10T17:17:13.680-08:00Thank you!Hello! I've been missing for a while (read as "not taking science classes"). This semester I will be back with Stem Cell Biology.<br />
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I've just discovered that this blog has rocketed in pageviews over the course of the past several months. I'm not sure what happened, but at least half of my total pageviews have come from Belgium. Not sure who's reading up on science, but I certainly hope it helps! It's very encouraging to know people are looking at what I post, since the purpose of this <i>is</i> to try and share science in a more understandable and explained way than the condensed jargon-dense speech of textbooks.<br />
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Cheers!Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-68003028510354521852012-05-10T15:14:00.005-07:002012-05-14T19:28:19.876-07:00Development Biology Exam 3 (Final but not comprehensive)<br />
http://dl.dropbox.com/u/22080433/3777_001.pdf<br />
http://dl.dropbox.com/u/22080433/3778_001.pdf<br />
http://dl.dropbox.com/u/22080433/4148_001.pdf<br />
http://dl.dropbox.com/u/22080433/4467_001.pdf <br />
<br />
And don't forget to check out fly battle vids:<br />
http://www.youtube.com/watch?v=ia57rw7PE_8<br />
http://www.youtube.com/watch?v=4pDU-cqvKJc<br />
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and fly sex vids:<br />
http://www.youtube.com/watch?v=zXXqQ2zJVMA<br />
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*5/13/12 edit: Thanks to Nadav who pointed out this nuance: stem cells can renew indefinitely, and progenitor cells can also self-renew, but only for a few more rounds.<br />
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*5/14/12 edit: More for future sake, it's not unoplakin but uroplakin in 4/13/12 lecture (which makes so much more sense i.e. ureter and all that) AND for 4/18/12 lecture, I wrote in for bone marrow transplantation that one needs mice clones, but actually that's not necessary. Using naked mice means they don't have an immune system to reject transplants anyway. The reason that's difficult in humans is you can't irradiate a human to ablate leukocytes (I mean you could, but who'd let you?)Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com2tag:blogger.com,1999:blog-6435239624608981366.post-75755938607516294122012-04-06T20:10:00.006-07:002012-04-08T19:52:24.015-07:00Dev Bio Exam 2 NotesPart 1<br />
http://dl.dropbox.com/u/22080433/3214_001.pdf<br />
Part 2<br />
http://dl.dropbox.com/u/22080433/3215_001-1.pdf<br />
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Notes from 3/16/12 (when I was sick, so these are neat notes courtesy of Angela Hsieh)<br />
http://dl.dropbox.com/u/22080433/3218_001.pdf <br />
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Part 3<br />
http://dl.dropbox.com/u/22080433/3216_001.pdf<br />
Part 4<br />
http://dl.dropbox.com/u/22080433/3219_001.pdf<br />
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The scanner and I had a few more spats, so if you can't read anything, leave a comment or email/fbook me and I'll translate :D<br />
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****4/8/12 UPDATE<br />
Edit to notes from 3/9/12: nodal is expressed in LPM not "central plate mesoderm."<br />
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Edit to last diagram from 4/6/12: the yellow boxes or "sclerotome" split and rejoin, not the blue ovals of the myotome.<br />
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Edit to description of trunk NCCs from 3/14/12: trunk NCC migration in the dorsolateral direction is inhibited by both slit and ephrin. At first glance, ephrin as a repulsor may seem counterintuitive since ephrin is a cell-cell <i>adhesion</i> molecule. But as it turns out, ephrin to ephrin receptor binding can result in both adhesion and repulsion, depending on relative levels of expression of both molecules (the details of which are beyond of the scope of lecture).<br />
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Many thanks to Nadav Nahumi for pointing out these errors. If anyone else spots some, please notify me.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com3tag:blogger.com,1999:blog-6435239624608981366.post-10008834562600757882012-02-27T17:00:00.002-08:002012-02-28T17:52:33.818-08:00In preparation for DevBio exam 1 in two days!!! Our professor talks really fast (despite being interesting at the same time) so I provide these transcribed notes in case you are missing something. And you now have something to follow with the podcasts. :D<br />
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Because I was squabbling with the scanner, I had to scan it in 4 parts. The first part being the first page.....yeah.. Enjoy! <br />
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DevBio Exam 1 teaser (a.k.a. page 1): http://dl.dropbox.com/u/22080433/devbioexam1pg1.pdf<br />
DevBio Exam 1 section 2: http://dl.dropbox.com/u/22080433/devbioexam1section2.pdf<br />
DevBio Exam 1 section 3: http://dl.dropbox.com/u/22080433/devbioexam1section3.pdf<br />
DevBio Exam 1 section 4: http://dl.dropbox.com/u/22080433/devbioexam1section4.pdf<br />
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Note: there are a few Q's on the side panels--you can ignore some of those. I pestered our professor with those, and they are mostly out of scope of the lecture he said.Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com1tag:blogger.com,1999:blog-6435239624608981366.post-48357696209080361172012-02-18T20:40:00.000-08:002012-09-19T13:29:07.464-07:00Return of the Unofficial TA!Hello all,<br />
<br />
I'm back this semester with Development Biology (while attempting to study for MCATs the same semester). The given textbook is dense and difficult to read, but our professor does cover a great deal in lecture, so I will be posting my lecture notes (there will also be lots of drawings!!!). If there is demand for a study guide or review session, let me know (either you know how to find me, or leave a comment).<br />
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Happy studying.<br />
<br />Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-60013124259189956942011-06-08T15:40:00.000-07:002011-06-08T15:40:04.546-07:00Fall 2010's Biochemistry NotesLinks:<br />
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exam 2<br />
https://docs.google.com/document/d/1v-8292bbokdMypiTyumz0QJ9CW0ScJdeyyCSzgNGTOY/edit?hl=en_US&authkey=CL67zqUE<br />
<br />
exam 3<br />
https://docs.google.com/leaf?id=0B99-sSwVe231YWRmYzEzOWItNmNiOS00ZWQzLTgxODYtZjhiOWI3ZWYzOGQ1&hl=en_US&authkey=CPf78pwM<br />
<br />
exam 4<br />
https://docs.google.com/leaf?id=0B99-sSwVe231YjY1ZWQyMjctZjFmZS00MDBkLTkyNDgtZDFiYzA1YmJlMmQy&hl=en_US&authkey=CJjumagBAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-79566216312303052732011-05-18T13:29:00.000-07:002011-05-18T13:29:02.134-07:00Japanese Cartoons in BiologyRemember Sonic Hedgehog protein? Well, how about the Raichu probe?<br />
<br />
Raichu stands for <b><span class="Apple-style-span" style="color: red;">Ra</span></b>s <b><span class="Apple-style-span" style="color: red;">i</span></b>nteracting <span class="Apple-style-span" style="color: red;"><b>ch</b></span>imeric <b><span class="Apple-style-span" style="color: red;">u</span></b>nit, where someone has attached a CFP to Raf to make a chimera protein (fusion protein). Then, this Raf-CFP can can interact with Ras engineered with YFP. Normally, Raf only binds to Ras-GTP, not Ras-GDP. Bioengineers invented Raichu to be able to detect and localize Ras-GTP in action, using FRET. When they use fluorescence to zap the cell with CFP's excitation wavelength, CFP is excited and sends out light at the CFP emission wavelength. Conveniently, CFP's emission wavelength is YFP's excitation wavelength, so if Ras-CFP has a GTP and binds Raf-YFP, then your initial light excites CFP which excites YFP so you will see the color of YFP's emission, not CFP's emission. If you see that, that means Ras is bound very closely to Raf.<br />
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Raichu actually stands for Ras interacting <i>protein</i> chimeric unit, but it's not surprising they left out the "p" when making the acronym. :PAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-13826339285191024252011-05-18T07:52:00.001-07:002011-05-18T07:52:48.263-07:00Apoptosis videoNice video for apoptosis, covers and LABELS all the proteins we learned about:<br />
<br />
http://www.youtube.com/watch?v=hqhxnWty5jcAnuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-4394649573416259772011-05-17T13:15:00.000-07:002011-05-17T13:15:14.514-07:00Book Notes: Cancer Part 2<ul><li><span class="Apple-style-span" style="color: red;"><b>cancer critical genes</b><span class="Apple-style-span" style="color: black;">: small subset of genes whose mutation usually causes cancer</span></span></li>
<ul><li><span class="Apple-style-span" style="color: red;"><span class="Apple-style-span" style="color: black;"><b>proto-oncogenes</b>: genes who, when mutant, are overactive and drive cells to become cancerous</span></span></li>
<li><span class="Apple-style-span" style="color: red;"><span class="Apple-style-span" style="color: black;"><b>tumor suppressor genes</b>: genes who prevent cancerous cells (when mutant, they are inactive and allow cells to become cancerous)</span></span></li>
<li><b>DNA maintenance genes</b>: genes who, when mutant, are inactive and allow cells to become genomically unstable, which may or may not lead to cancer</li>
</ul><li>to test possible proto-oncogenes, you insert a copy of the oncogene (mutated version)</li>
<ul><li>if cells become cancerous, then the inserted gene really was an oncogene</li>
</ul><li>to test possible tumor suppressor genes, you have to knockout both copies of the gene in the cell</li>
<ul><li>as long as cells have one copy of a tumor suppressor gene, they can withhold the cell from abnormal growth</li>
</ul><li><b><span class="Apple-style-span" style="color: orange;">tumor viruses</span><span class="Apple-style-span" style="font-weight: normal;">: viruses who don't intentionally cause cancer, but by inserting their own genome into the DNA, causes a mutation that leads to cancer</span></b></li>
<ul><li><b><span class="Apple-style-span" style="font-weight: normal;">they are usually retroviruses, who carry RNA that is reverse transcribed into DNA and then inserted</span></b></li>
</ul><li>DNA decoding of transformed cells helped determine oncogenes</li>
<ul><li>tumor DNA is fragmented and put with fibroblasts that already divide a lot (but are still normal)</li>
<li>if one of the fragment contains an oncogene, some of the cells will start forming masses: transformed!</li>
<li>sequencing the genome of these transformed cells compared to the non transformed cells showed the oncogene</li>
<li>Ras was the first, and is mutated in 1 of every 5 cancers</li>
<ul><li>a point mutation prevents it from hydrolyzing GTP, so it is hyperactive and never shuts off</li>
<li><b><i>dominant effect</i><span class="Apple-style-span" style="font-weight: normal;">: only one copy of the Ras gene needs to be mutated this way in order to produce an uncontrolled effect</span></b></li>
</ul></ul><li>studying retinoblastoma, eye cancer, helped decode tumor suppressor genes</li>
<ul><li>there were 2 types of eye cancer, one hereditary, one just random. The hereditary version impacted both eyes (so it must be a genomic problem in all the eye cells)</li>
<li>they found a deletion on chromosome 13, where one copy of the Rb protein was missing</li>
<li>it's hereditary because families inherit the one good copy and one bad copy, so as soon as the one good copy is mutated, they get retinoblastoma</li>
<li>the non hereditary is random and usually in one eye because there must be 2 separate events that mutate both copies of Rb in one cell</li>
<li>Rb regulates cell cycle, so mutated Rb allows inappropriate and uncontrolled cell proliferation</li>
</ul><li>usually tumor suppressor genes are identified by screening many cancers and finding out what gene is repeatedly missing or defective</li>
<li>tumor suppressor genes can be inactivated in many ways</li>
<ul><li>chromosome deletion, point mutation, segregation/recombination errors, etc, etc</li>
<li><b>loss of heterozygosity</b>: where one copy of a cancer-critical gene is lost or deactivated, so now the cell has a <i>predisposition</i> to cancer (being cancer-prone)</li>
<li>epigenetics: methylation of a tumor suppressor gene causes it to be stored in heterochromatin and silenced for all the progeny</li>
</ul><li>proto-oncogenes can be overactivated in many ways</li>
<ul><li>point mutation, deletion, gene amplification, chromosomal rearrangement that ends up altering a gene or the regulatory sequence of the gene</li>
</ul><li>3 methods of cancer-critical gene identification</li>
<ol><li><span class="Apple-style-span" style="color: #6aa84f;">CGH (comparative genomic hybridization)</span>: DNA fragments from normal cells and from cancers cells are fluorescently labelled</li>
<ul><li>the labelled fragments hybridize to complementary sequences on a DNA microarray (where each spot corresponds to a known sequence of the genome)</li>
<li>where the tumor complement binds, it means that many copies of the gene exist (gene amplification)</li>
<li>where the normal complement binds, it means there was a deletion</li>
<li>where there is no light, it means that the two copies of the genome exist and are paired up (blocking any probes from hybridizing)</li>
</ul><li>DNA microarray can also be used with mRNA probes</li>
<li>RNAi: siRNA can be engineered to knockout a specific gene by destroying the mRNA of that gene or inhibiting the translation</li>
</ol><ul><ul><li>then you observe whether the knockout cell becomes cancerous or not</li>
</ul><li>best test involves doing knockouts or causing overexpression of a gene in actual mice</li>
<ul><li>mice that develop tumors soon after prove the possibility of that gene being a cancer-critical gene</li>
</ul></ul><li>so what are the pathways in which these cancer-critical genes actually cause cancer?</li>
<ol><li>cell proliferation pathways: just about any and every signaling protein can be affected, usually with proteins that activate or relay signals becoming hyperactive (proto-oncogenes) -- i.e. Ras, EGF receptor</li>
<ul><li>the inhibitors involved in pathways tend to be the tumor suppressor genes -- i.e. Rb</li>
<ul><li>cancer cells might eliminate Rb, or by inhibiting the inhibitor of the inhibitor of Rb (in times of stress, p16 blocks cyclinD/Cdk4 from inhibiting Rb, who inhibits E2F from continuing the cell cycle, SO, if a mutation inhibits p16, cyclinD/Cdk4 is free to inhibit Rb so that E2F happily progresses the cycle)</li>
</ul></ul><li>cell growth pathways: cancer needs to be able to grow uncontrollably, otherwise uncontrolled division will just lead to smaller and smaller cells</li>
<ul><li>PI3/Akt pathway important for growth, cancers activate this pathway in the absence of growth signals so they just grow all the time</li>
<ul><li>overactive Akt helps with protein synthesis, glucose uptake, and lipid synthesis</li>
<li>so it's usually PTEN phosphatase who is messed up, because he's the inhibitor of Akt</li>
</ul></ul><li>apoptosis pathways: apoptosis usually activates in response to DNA damage and mutations, so because cancers need mutations, they must shut off apoptosis so they can keep proliferating</li>
<ul><li>Bcl2 is a protein that normally inhibits apoptosis</li>
<ul><li>in a B-lymphocyte cancer, the Bcl2 gene is translocated to an active promoter, so Bcl2 is overexpressed</li>
<li>this keeps apoptosis from activating</li>
</ul></ul><li>DNA damage pathways: but really the star player is <span class="Apple-style-span" style="color: #3d85c6;"><b>p53</b></span></li>
<ul><li>known as the guardian angel gene, p53 is super critical because it happens to be involved in 2 of the above 3 pathways, and in DNA damage systems</li>
<ul><li>to start, p53 is normally kept at very low levels: continuously made and continuously degraded</li>
<li>when a cell is stressed or the genome is damaged, p53 levels shoot up</li>
<li>active p53 (1) stops any cell dividing to buy some time (2) activates DNA repair systems (3) or activates apoptosis when nothing can be done</li>
<li>this is the protection that prevents cells with just a few mutations from blowing over into cancer</li>
<li>p53 is a gene regulatory protein, it activates expression of other proteins that carry out the above-listed functions</li>
<li>mutation of p53 usually happens in its DNA-binding domains, so it can't bind to genes and activate transcription, and the cell can (1) divide uncontrollably (2) avoid apoptosis (3) have genetic instability w/o DNA repair and (4) ignore stresses like radiation and drug treatment</li>
</ul></ul></ol><li>DNA tumor viruses are the lysogenic viruses, who integrate their genome into the host genome and sit quietly</li>
<ul><li>virus genome contains stuff that take control of reproduction machinery to generate more viruses</li>
<li>accidents can occur that activate some components of the virus genome that activate DNA replication and division, leading to tumor formation</li>
<li>e.g. papillomavirus produces a protein E7 that blocks Rb, and an E6 protein that inhibits p53</li>
</ul><li>Metastasis has 2 steps: invasion and re-establishment</li>
<ul><li><span class="Apple-style-span" style="color: #674ea7;"><b>EMT (epithelial to mesenchymal transition)</b><span class="Apple-style-span" style="color: black;">: normal organism development process where epithelial cells change from adhesive and stable to less adhesive and migratory</span></span></li>
<ul><li><span class="Apple-style-span" style="color: #674ea7;"><span class="Apple-style-span" style="color: black;">this happens when E-cadherin is lost and cells can't form junctions, so they can go out exploring and getting into blood vessels</span></span></li>
</ul><li>colonization: cancer cells must acquire the ability to proliferate in a new environment in order to do this</li>
<ul><li>that's why most cancer cells that escape the primary tumor don't actually form new metastases</li>
</ul></ul><li><span class="Apple-style-span" style="color: #a64d79;"><b><i><u>CASE STUDY: COLORECTAL CANCER</u></i></b></span></li>
<li>colon epithelium has rapid turnover rate, so it is constantly growing and dividing (at a controlled rate) to replace old cells</li>
<ul><li>renewal depends on special <span class="Apple-style-span" style="color: #cc0000;">crypts</span> (pockets) of stem cells in the epithelium</li>
<li>benign tumors look like small protruding masses called polyps, the first sign of abnormal growth</li>
<li>the most common mutations happen to 3 proteins: <span class="Apple-style-span" style="color: #e06666;"><b>K-Ras, p53, Apc</b></span></li>
<ul><li>Apc is an inhibitor of the Wnt pathway but causing degradation of B-catenin (recall B-catenin can go in to the nucleus to activate transcription)</li>
<li>but tumors tend to form faster with a hyperactive B-catenin rather than an inactive APC</li>
<li>the high frequency of APC mutations comes from APC being involved in other pathways as well and getting mutated affects the mitotic spindle formation, so chromosome segregation is messed up</li>
</ul></ul><li>one form of colon cancer is hereditary, due to a mutation in DNA mismatch repair</li>
<ul><li>the inherited predisposition involves having one messed up copy of the repair gene and one good copy</li>
<li>once a mutation inactivates the one good copy, cells will start building mutations, increasing chances for cancer</li>
</ul><li>tumors often have specific order of mutations</li>
<ul><li>in most cases, Apc is one of the first to go</li>
<ul><li>mutations w/o Apc's guardianship will build up</li>
</ul><li>then Ras falls apart</li>
<ul><li>cells now proliferate w/o substrate or neighbor inhibition</li>
</ul><li>then p53 is messed up</li>
<ul><li>now cells can mutate faster and avoid apoptosis</li>
</ul><li>each tumor will mutate its own variation of similar genes (like messing up the Ras regulator as opposed to Ras itself), but the control mechanisms need to be disabled in a proper order</li>
<li>even different patients with the same type of cancer will have different mutations, which makes cancer treatment very difficult</li>
</ul><li>cancer prevention or early diagnosis of a primary tumor is the best method of avoiding cancer</li>
<ul><li>treatment is difficult because you must remove ALL the bad cells, or else even just one cancer stem cell will regenerate a tumor</li>
<li>surgery makes it difficult to get every tiny metastasis, and drug/chemical treatments also hurt good cells</li>
</ul><li>traditional cancer treatment exploits genetic instability</li>
<ul><li>radiation and drugs kill cancer cells because they can't repair the DNA damage, while normal cells will pause cycling (so you lose hair) but will fix the damage</li>
<li>unfortunately, cancer cells can evolve resistance to treatment (like bacteria!)</li>
</ul><li>new treatments exploits CAUSE of genetic instability</li>
<ul><li>first, consider that there are multiple ways to repair DNA damage</li>
<ul><li>a single strand break is fixed by one system</li>
<li>if that system is down, the stalled replication fork that results when DNA Pol encounters the ssbreak can be fixed by homologous recombination</li>
</ul><li>second, consider that cancer cells tend to lose some of the repair systems in order to become cancer</li>
<ul><li>for example, let's say a cancer cell can't repair a stalled fork, but it can fix ssbreaks</li>
</ul><li>third, consider that we can devise drugs to impair repair systems</li>
<ul><li>in the example with the cancer cell that can't repair stalled forks, what if we give it a drug that inhibits the repair of ssbreaks?</li>
<li>now the tumor can't repair ssbreaks, and it already couldn't repair stalled forks, so it will die....</li>
<li>BUT the normal cells live, because unlike the cancer cell, they CAN repair stalled forks, even if the drug also affects them and inhibits the repair of ssbreaks</li>
</ul><li>these treatments take advantage of the fact that normal cells have multiple options, while cancer cells have very few repair/survival options because of their mutations</li>
</ul><li>cancer cells have still another advantage: genomic heterogeneity</li>
<ul><li>even though they are all descended from the same ancestor, they have such a high rate of mutation that much of the population have different variations in their genome</li>
<li>this means you can't just use one type of treatment to eradicate a whole tumor</li>
<li><b>multidrug resistance</b>: phenomenon where a cell, after surviving just one drug treatment, somehow is resistant to a bunch of other drugs that it's never met before</li>
<ul><li>this happens when a gene called <b><span class="Apple-style-span" style="color: #cc0000;">Mdr1</span></b> is amplified (has multiple copies now)</li>
<li>Mdr1 makes a transporter that pumps out drugs, overexpression of the protein makes sure no drugs hang around and damage the cell: they just get pumped out</li>
</ul></ul><li>new therapies are being developed due to our increasing knowledge</li>
<ul><li>targeting the one hyperactive protein that a cancer may be dependent upon (oncogene addiction: the cell is addicted to/dependent on the oncogene)</li>
<li>send in antibodies that either carry toxins or enzymes that cleave pro-drugs into toxins at the cancer site (because antibodies bind so specifically, you use anti-cancer antibodies)</li>
</ul><li>we can also engineer small molecules that target the one hyperactive protein of oncogene addiction</li>
<ul><li>e.g. CML (chronic myelogenous leukemia) is associated with Philadelphia chromosome</li>
<li>this chromosome snaps the genes <span class="Apple-style-span" style="color: red;">Abl </span>and<span class="Apple-style-span" style="color: red;"> Bcr</span> and rejoins them so they fuse into Bcr-Abl</li>
<li>the fusion protein combines the Tyr kinase of Abl and the active expression of Bcr to make a hyperactive kinase</li>
<li>this prevents apoptosis of unnecessary lymphocytes, so they build up in the blood</li>
<li>a special molecule called <span class="Apple-style-span" style="color: #e69138;"><b>Gleevec</b><span class="Apple-style-span" style="color: black;"> blocks Bcr-Abl, and effectively eliminates CML in early diagnosis patients</span></span></li>
<li><span class="Apple-style-span" style="color: #e69138;"><span class="Apple-style-span" style="color: black;">less effective with later patients b/c cancers have usually evolved resistance to Gleevec binding</span></span></li>
<ul><li><span class="Apple-style-span" style="color: #e69138;"><span class="Apple-style-span" style="color: black;">probably going to have to do a cocktail to fully eradicate cancerous cells</span></span></li>
</ul></ul><li>another approach is to target the tumor blood vessels</li>
<ul><li>there are drugs that inhibit the VEGF receptor</li>
<li>also, endothelial cells that are about to make new blood vessels have distinguishable characteristics that we can engineer molecules to attack them specifically</li>
</ul><li>the final method is to enhance the existing immune system to target tumors</li>
<ul><li>we can tag cancer cells with antibodies to attract macrophages to gobble them up</li>
</ul><li>the future of treatment is probably going to involve a simultaneous combinations of drugs and molecules, because no cell will have resistance to all of them without some time to evolve (i.e. if we used the drugs one by one, every time, there would always be some cells resistant the single drug used)</li>
</ul><br />
<br />
pg. 1230 - 1240Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-41893477099454356442011-05-17T06:58:00.000-07:002011-05-17T06:58:52.927-07:00Book Notes: Cancer Part 1<ul><li>normal cells work cooperatively, and obey signals and controls on division and growth so the organism can survive</li>
<ul><li>cancer cells work "selfishly", growing and dividing out of control, nabbing up nutrients and creating blood vessels so they can survive</li>
<li>know the difference between BENIGN and MALIGNANT (is it invasive)</li>
</ul><li>several types of cancer by tissue of origin</li>
<ul><li><span class="Apple-style-span" style="color: #cc0000;"><b>carcinoma</b><span class="Apple-style-span" style="color: black;">: from epithelium, most common because epithelium is usually exposed to stresses and dangers (serves as protective barrier, making it vulnerable to damage and mutation)</span></span></li>
<li><span class="Apple-style-span" style="color: #cc0000;"><b>sarcoma</b><span class="Apple-style-span" style="color: black;">: from muscle/connective tissue</span></span></li>
<li><span class="Apple-style-span" style="color: #cc0000;"><b>leukemia/lymphoma</b><span class="Apple-style-span" style="color: black;">: from white blood cells, lymphocytes, and nerve cells</span></span></li>
</ul><li>cancer cells tend to be all clones of the primary tumor</li>
<ul><li>patients with CML, a leukemia with the mutation Philadelphia chromosome, show that the chromosome break happens at exactly the same place for all cells in the tumor, but it happens a few hundred basepairs different between patients</li>
<li>this implies cells of one tumor all derive from one ancestral cancerous cell and are all clones</li>
</ul><li>cancer results from a genetic mutation, which may be from chemical carcinogens or radiation/UV light</li>
<ul><li>however, you need more than one mutation in ONE CELL LINE to have cancer</li>
<li>organisms have a high overall rate of mutation, but that rate is including all the cells of a human (there are several trillion cells in the body, a human has about several trillion mutations over the lifetime, so it's like maybe one mutation per cell per day, and remember that repair machinery usually fixes the mutation)</li>
<li>cancer usually happens in old age because that's when you've likely built up a lot of mutations and the tumor has had some time to grow into a visible mass</li>
<ul><li>e.g. leukemia didn't show up until 5 years after the atomic bombs razed Hiroshima, and lung cancer takes 20 years of heavy smoking before surprise!</li>
</ul><li>rule of thumb: minimum 5 mutations to become cancerous</li>
<li>creating a mutant cancerous cell takes microevolution</li>
<ul><li>mutation rate: how fast does this kind of cell get mutations?</li>
<li>number of reproducing individuals: how many cells in this tissue divide at a time?</li>
<li>rate of reproduction: how fast can a cell divide and how many times can it divide?</li>
<li>selective advantage: does the mutation kill the cell or give it a bonus or do nothing?</li>
<li>you need several rounds of division (epithelial cells divide constantly) with <span class="Apple-style-span" style="color: #e69138;">advantageous</span> mutations carrying through each step until you shake loose the body's regulatory systems</li>
</ul></ul><li>cancer also involves epigenetic mutations</li>
<ul><li>epigenetics involving histone modification is a way of controlling which genes are active and which are silent</li>
<li>mutations happen with the enzymes that modify histones, the proteins that interpret the histone code, etc et</li>
<li>these mutations are also passed on to daughter cells</li>
</ul><li>because cancer cells proliferate uncontrollably, they are also genetically <span class="Apple-style-span" style="color: #f1c232;">unstable</span></li>
<ul><li>it's usually because the cancers have a mutation in DNA repair/maintenance genes</li>
<li>the increase in mutation rate plus their rapid division rate makes their evolution rate really fast--cancers with genetic instability speed rapidly towards malignancy</li>
<li>cancer cells can also accumulate bad mutations that end up killing it, so it is the cancer cell with the right mutations that will persist into tumors and metastases</li>
</ul><li>the core mutations of cancer cells tend to be in control of cell death, control of cell differentiation, or both (for optimal cancer growth :D )</li>
<ul><li>any mutation or external condition that promotes cell growth can and will help cancerous cells</li>
<li>normal tissue growth controlled by apoptosis, especially when apoptosis serves as a control to destroy mutated cells</li>
<ul><li>cells that don't do apoptosis when they're supposed to keep growing and passing on mutations, potentially forming cancer</li>
</ul><li>stem cells will produce daughter cells that proliferate for a bit before committing to differentiation</li>
<ul><li>if the differentiation step is blocked, daughter cells just keep dividing and dividing</li>
</ul></ul><li>other core mutations are DNA damage response and other stress responses</li>
<ul><li>the DNA damage response goes hand in hand with the cell cycle checkpoints: mutations in either system won't be able to stop a damaged cell from continuing to divide and pass on the mutations</li>
<li>being able to survive through these mutations makes them more undefeatable</li>
</ul><li>the final barrier to cancers is <span class="Apple-style-span" style="color: #6aa84f;">replicative cell senescence</span>, when the telomeres run out, the cell would normally do apoptosis</li>
<ul><li>cancers can avoid the chkpt when telomeres run out and just keep dividing without telomeres (meaning every round shrinks the chromosome by a bit)</li>
<li>cancers can also have a mutation that reactivates telomerase or a mutation that creates something similar to telomerase</li>
</ul><li>new theory suggests tumors are organized with <span class="Apple-style-span" style="color: #3d85c6;">cancer stem cells</span> at the top and limited dividing lower cancerous daughter cells</li>
<ul><li>if the regular cancer daughter cell is implanted in a mouse, it can't generate a new tumor because it has limited dividing capacity</li>
<li>only a small percent of cells in a tumor (<1) can propagate indefinitely</li>
</ul><li>where do cancer stem cells come from?</li>
<ul><li>(1) from real stem cells and (2) from normal proliferating cells that developed a mutation that makes them propagate indefinitely like a stem cell</li>
<li>cancer stem cells divide slowly, so treatments that target rapidly dividing cells won't harm them, and then the tumor mass will regrow</li>
</ul><li>metastasizing cancer is the most dangerous, but it requires cancer cells to overcome some barriers first</li>
<ul><li>1st, the tumor cells must invade neighboring normal tissue and keep spreading</li>
<li>2nd, the tumor cells must find a blood or lymphatic vessel and get in</li>
<li>3rd, the tumor cells must grab onto a new site while floating through the vessel and form a small clump (<span class="Apple-style-span" style="color: #8e7cc3;">micrometastases)</span></li>
<li>4th, the clump must develop into a large tumor for stability</li>
<li>many cells are usually able to get into the vessel, but very few can attach and colonize in a new location</li>
<ul><li>even forming micrometastases is no guarantee of continued survival</li>
<li>metastasis usually requires a LOT of mutations in all the right places</li>
</ul></ul><li>a large tumor also needs a supply of nutrients</li>
<ul><li>a tumor over 1-2 mm will need to induce <span class="Apple-style-span" style="color: #a64d79;">angiogenesis</span>: formation of new blood vessel</li>
<li>normal and tumor cells secrete angiogenic signals in response to hypoxia (lack of nutrients and oxygen)</li>
<li>these signals activate transcription and secretion of VEGF</li>
<li>VEGF attract endothelial cells and stimulate growth of a blood vessel</li>
<li>induced vessels are usually disorganized and go random places with dead ends</li>
<ul><li>this leaves a lot of area still in hypoxia, which causes natural selection for cells that survive in tough conditions, which makes cancer even tougher to eradicate</li>
</ul></ul><li><span class="Apple-style-span" style="color: #cc0000;">stroma</span>: the surrounding connective tissue, even tumor cells talk with them like normal cells</li>
<ul><li>stroma contains fibroblasts, white blood cells, etc, for support</li>
<li>cancer cells send signals to the stroma</li>
<li>the stroma responds with signals that stimulate growth and secrete proteases to loosen the ECM for invasion</li>
<ul><li>tumor and stroma evolve together (experiment where a tumor is plucked out and put next to normal fibroblasts showed the tumor can't survive)</li>
<li>possible source of treatment: inhibit deranged stroma to kill tumors</li>
</ul></ul><li>sum up of characteristic cancer behaviors:</li>
<ol><li>survive and proliferate in weird conditions (not attached to substrate, etc, etc)</li>
<li>insensitive to anti-proliferation signals</li>
<li>avoid apoptosis</li>
<li>avoid stress and damage responses</li>
<li>induce help from stroma</li>
<li>induce angiogenesis</li>
<li>invade and proliferate far far away</li>
<li>genetically unstable</li>
<li>have stable telomeres</li>
</ol></ul><br />
<br />
pg. 1205 - 1223Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-58415271549326057522011-05-16T19:43:00.000-07:002011-05-16T19:43:08.442-07:00Book Notes: Signaling Part 5<ul><li>pathways involving gene regulatory proteins (who activate gene expression) depend on regulated proteolysis to control them</li>
<ul><li>Notch, Wnt, and Hedgehog are all these pathways, and they are especially involved in animal development</li>
</ul><li><span class="Apple-style-span" style="color: #cc0000;">Notch</span> is famous for affecting neural cell development</li>
<ul><li><b>lateral inhibition</b>: if one cell develops into a neuron, it signals to its neighbors to NOT being nerve cells and instead become epidermal</li>
<li>this occurs because the developing neuron displays the <span class="Apple-style-span" style="color: #cc0000;">Delta</span> signal protein on its surface</li>
<li>neighbors all have Notch receptors that bind Delta and receive the signal to develop into epidermis</li>
<li>Notch signaling can also be used for promoting neighbors to become the same cell type in other cases</li>
</ul><li>when Notch binds Delta, a membrane-bound protease cleaves Notch's tail</li>
<ul><li>the tail zooms off into the nucleus and activates a set of genes by binding and converting a transcriptional repressor into an activator</li>
<li>Notch is actually cleaved 3 times in its lifetime: (1) during synthesis, it is cleaved by Golgi to form heterodimer, (2) during ligand-binding, when the extracellular head is cleave off, and (3) after ligand-binding, when the cytoplasmic tail is cleaved off</li>
<li>cleavage is irreversible, meaning Notch is a one-time use receptor</li>
</ul><li>Notch and Delta are glycoproteins, and their specific match-up depends on how their sugar sidechains are modified (glycosylation is important!)</li>
<li><span class="Apple-style-span" style="color: #e69138;">Wnt</span> is famous for its versatility: it plays a role in catenins (i.e. jxns), cell polarity, etc, etc</li>
<ul><li>Wnt has 3 well-known pathways:</li>
<ol><li><span class="Apple-style-span" style="color: #f1c232;">Wnt/beta-catenin pathway</span>: Wnt affects the gene regulatory protein beta-catenin</li>
<li><span class="Apple-style-span" style="color: #f1c232;">planar polarity pathway</span>: coordinate polarization in plane of epithelium</li>
<li><span class="Apple-style-span" style="color: #f1c232;">Wnt/Ca2+ pathway</span>: stimulates increase of calcium level</li>
</ol><li>Wnt is a secreted molecule that binds to <span class="Apple-style-span" style="color: #f6b26b;">Frizzled</span> receptors</li>
<ul><li>Wnt-bound Frizzled recruits <span class="Apple-style-span" style="color: #f6b26b;">Dishevelled</span>, which then relays the signal down one of the 3 pathways</li>
</ul></ul><li>for the beta-catenin pathway, Wnt binds to both Frizzled and an <b>LRP (LDL-receptor-related protein)</b></li>
<ul><li>any beta-catenins not involved in jxn-forming is bound by <b>degradation complex</b>, which keeps it away from the nucleus and promotes its degradation</li>
<li>2 kinases (GSK3 and CKIgamma) phosphorylate C tail of LRP, which inactivates axin (part of the degradation complex), so free beta-catenin accumulates and migrates to the nucleus</li>
</ul><li>without Wnt, the set of genes controlled by Wnt are silent</li>
<ul><li>LEF1/TCF proteins bound to Groucho (a co-repressor)</li>
<li>when Wnt activates, beta-catenin comes in and displaces Groucho as the co-partner</li>
<li>Beta-catenin serves as a co-ACTIVATOR for the genes</li>
<li>c-Myc is one of the proteins expressed by Wnt signaling, and it stimulates growth and proliferation (potential cancer!!!)</li>
</ul><li><span class="Apple-style-span" style="color: #6aa84f;">Hedgehog</span> is similar to Wnt: is secreted, modified by lipids, trigger switch from repression to activation, and leads to growth/proliferation response</li>
<ul><li>3 transmembrane proteins mediate responses to the Hedgehog proteins:</li>
<ul><li><span class="Apple-style-span" style="color: #38761d;">Patched</span>: multipass with some of it in intracellular vesicles, and some on the cell surface to bind Hedgehog</li>
<li><span class="Apple-style-span" style="color: #38761d;">Smoothened</span>: 7pass transmembrane</li>
<li><span class="Apple-style-span" style="color: #38761d;">iHog</span>: 4-5 immunoglobulin domains and 2-3 fibronectin domains on cell surface, serve as co-receptors with Patched</li>
<li>Patched normally sequesters Smoothened </li>
<li>Hedgehog binding to iHog partnered with Patched inhibits Patched by causing endocytosis and degradation of Patched</li>
<ul><li>result is that Smoothened is free and phosphorylated and continues the signaling pathway</li>
</ul></ul><li><span class="Apple-style-span" style="color: #45818e;">Cubitus interruptus (Ci)</span>: gene regulatory protein that is a target of Hedgehog</li>
<ul><li>in normal conditions, Ci is proteolysed into smaller proteins that hang in the nucleus as repressors</li>
<li>this proteolysis is dependent on phosphorylation by PKA and GSK3 and CK1 (all Ser/Thr kinases)</li>
<li>when Hedgehog binds, Smoothened runs free and recruits <span class="Apple-style-span" style="color: #3d85c6;">Costal2</span>, a scaffold protein, and <span class="Apple-style-span" style="color: #3d85c6;">Fused</span>, another Ser/Thr kinase</li>
<ul><li> Costal2 normally holds the proteolytic kinases together to process Ci, but binding to Smoothened blocks this ability</li>
</ul><li>uncleaved Ci also goes to the nucleus, but acts as an activator</li>
<li>uncleaved Ci can also express the Patched gene = more Patched inhibits further Hedgehog signaling</li>
</ul></ul></ul><br />
<br />
pg. 946 - 952Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0tag:blogger.com,1999:blog-6435239624608981366.post-11779761948006282852011-05-16T18:03:00.000-07:002011-05-18T13:16:33.541-07:00Book Notes: Signaling Part 4<ul><li>G proteins do more than play with cAMP and Ca2+</li>
<ul><li>the <b><i>a</i><span class="Apple-style-span" style="font-weight: normal;"> subunit of G12 activates GEF for the Rho GTPase</span></b></li>
<li><b><span class="Apple-style-span" style="font-weight: normal;">others bind and open/close ion channels to change membrane potential</span></b></li>
<li><b><span class="Apple-style-span" style="font-weight: normal;">others phosphorylate channels or change levels of cNMP (e.g. cAMP) that affect channels</span></b></li>
</ul><li>olfactory receptors utilize cAMP-gated ion channels</li>
<ul><li>odor particle binds to olfactory receptor</li>
<li>the receptor activates the G protein <b><span class="Apple-style-span" style="color: #cc0000;">Golf </span>(G subscript olf for olfactory)</b></li>
<li>Golf activates adenylyl cyclase = lots of cAMP</li>
<li>cAMP binds to cation channels that allow Na+ to flood in</li>
<li>neuron containing the olfactory receptor is depolarized and sends a potential down the axon to the brain</li>
<li>each neuron makes only 1 type of receptor that senses only a small set of odors</li>
</ul><li>visual receptors utilize cGMP-gated ion channels</li>
<ul><li>rod photoreceptors are cells with a rodlike outer and inner segment, followed by the body, followed by the axon and synapse</li>
<li>the outer segment looks like a pancake stack with lots of rhodopsin molecules in them</li>
<li>the plasma membrane coating the stacks have lots of cGMP-gated cation channels</li>
<li>in the dark, cGMP remains bound to the channels, keeping them open</li>
<li>in the light, the <i>cis </i>chromophore in the rhodopsin absorbs a photon and isomerizes into <i>trans</i></li>
<li>the changed chromophore causes the protein <span class="Apple-style-span" style="color: #e69138;">opsin</span> to change conformation and activates the G protein <span class="Apple-style-span" style="color: #bf9000;">transducin (Gt)</span></li>
<li>transducin <b><i>a </i><span class="Apple-style-span" style="font-weight: normal;">subunit activates cGMP PDE, which removes cGMP</span></b></li>
</ul><li><b><span class="Apple-style-span" style="font-weight: normal;">visual system resets itself VERY QUICKLY so that we can see the change in light in the next instant</span></b></li>
<ul><li><span class="Apple-style-span" style="color: #6aa84f;">rhodopsin kinase</span> phosphorylates the tail of activated rhodopsin, to prevent further activation of Gt</li>
<li><span class="Apple-style-span" style="color: #45818e;">arrestin</span> binds to Pi-rhodopsin to fully inhibit rhodopsin activity</li>
<li>RGS acts as GAP to make Gt hydrolyze the GTP</li>
<li>light also causes calcium channels to close, so calcium levels fall, so guanylyl cyclase hurries to make more cGMP, returning to normal "dark" mode</li>
<li>negative feedback allows system to adapt to continuous light, and be sensitive to CHANGES in light (seeing camera flash in broad daylight)</li>
</ul><li>all the steps of intracellular signaling pathways are potential<span class="Apple-style-span" style="color: #3d85c6;"> SIGNAL AMPLIFICATION</span> steps</li>
<ul><li>but for rapid sensing, cells must be able to both amplify a signal quickly and destroy the response just as quickly</li>
<li>therefore, any and every protein/molecule in the pathway can be a target for regulation</li>
</ul><li>GPCRs are usually desensitized in 3 ways</li>
<ul><li>receptor inactivation: receptor interaction with G protein is blocked</li>
<ul><li>e.g. <span class="Apple-style-span" style="color: #674ea7;"><b>GRK (GPCR kinase)</b><span class="Apple-style-span" style="color: black;"> like RK that phosphorylates GPCR after the receptor has been activated by ligand-binding</span></span></li>
<li><span class="Apple-style-span" style="color: #674ea7;"><span class="Apple-style-span" style="color: black;">then (as with rhodopsin) an inhibitor like arrestin binds and blocks the G protein interaction</span></span></li>
</ul><li>receptor sequestration: move receptor inside the cell to block access to ligand signal</li>
<ul><li><span class="Apple-style-span" style="color: #674ea7;"><span class="Apple-style-span" style="color: black;">arrestin can also couple the receptor to endocytosis machinery and clathrin</span></span></li>
<li><span class="Apple-style-span" style="color: #674ea7;"><span class="Apple-style-span" style="color: black;">later dephosphorylation returns the receptor to the surface</span></span></li>
</ul><li>receptor down-regulation: receptor is destroyed post-activation</li>
<ul><li>endocytosis of receptor in some cases lead to the receptor ubiquitylation and lysosomal degradation</li>
</ul></ul><li><span class="Apple-style-span" style="color: #a64d79;"><b>EZCR (enzyme-coupled receptor)</b><span class="Apple-style-span" style="color: black;">: transmembrane receptor with a cytosolic domain that is an enzyme or associates with another enzyme (note, the abbreviation is my own invention)</span></span></li>
<ul><li><span class="Apple-style-span" style="color: #a64d79;"><span class="Apple-style-span" style="color: black;">exist 6 classes of EZCRs</span></span></li>
<ol><li><span class="Apple-style-span" style="color: #cc0000;">receptor Tyr kinase</span>: phosphorylate Tyr residues on itself and some signaling proteins</li>
<li><span class="Apple-style-span" style="color: #cc0000;">Tyr kinase associated receptor</span>: recruit cytoplasmic Tyr kinase</li>
<li><span class="Apple-style-span" style="color: #cc0000;">receptor Ser/Thr kinase</span>: phosphorylate Ser/Thr on itself and some gene regulatory proteins</li>
<li><span class="Apple-style-span" style="color: #cc0000;">His kinase associated receptor</span>: phosphorylate itself on His and transfers the Pi to a signaling protein</li>
<li><span class="Apple-style-span" style="color: #cc0000;">receptor guanylyl cyclase</span>: catalyzes production of cGMP</li>
<li><span class="Apple-style-span" style="color: #cc0000;">receptorlike Tyr phosphatase</span>: remove Pi from Tyr of signaling proteins (ligands are unknown)</li>
</ol></ul><li><span class="Apple-style-span" style="color: #e69138;">RTK (receptor Tyr kinase)</span>: bind hormones like insulin, growth factors, and ephrins (cell-surface ligands as opposed to free floating signals)</li>
<ul><li>ephrins and <b>EPH</b>rin receptors both make responses in the receptor cell and the ligand cell: "<span class="Apple-style-span" style="color: #f6b26b;">bidirectional signaling</span>"</li>
<li>ligand binding causes RTKs to dimerize and cross-phosphorylate each other</li>
<li>the phosphorylated Tyr residues serve as docking sites for specific intracellular signaling proteins</li>
<li>these signaling proteins are either activated by docking or by receiving phosphorylation from the RTK</li>
<li>proteins dock by <b>modular interaction domains</b> (remember SH2, SH3, etc etc), sites of protein protein binding that don't affect function</li>
</ul><li>examples of docked proteins are <span class="Apple-style-span" style="color: #f1c232;">PI3-kinase</span> and <span class="Apple-style-span" style="color: #f1c232;">PLCgamma</span> (PLCbeta associates with GPCRs)</li>
<li>adaptor proteins with lots of modular domains connect docked proteins to other proteins that DON'T have these awesome domains</li>
<ul><li>example being <b><i><u><span class="Apple-style-span" style="color: #6aa84f;">RAS</span></u></i></b></li>
</ul><li>Ras and Rho families are the only monomeric GTPase families to relay signals from surface receptors</li>
<ul><li>as a family, they can coordinate one signal to many pathways</li>
<li>Ras has lipid groups to keep it anchored to the cytoplasmic leaflet of the membrane</li>
</ul><li>RTKs activate Ras through adaptors and Ras-GEF</li>
<ul><li>case study: fly eye is composed of <span class="Apple-style-span" style="color: #45818e;">ommatidia</span>, which are sets of 8 photoreceptor cells and 12 accessory cells</li>
<li>the <span class="Apple-style-span" style="color: #3d85c6;">Sevenless (Sev)</span> mutant gene causes flys to be blind in UV light, because mutant Sev prevents normal development of the R7 photoreceptor</li>
<li>BUT it turns out for some blind flies, the Sev gene is kept normal: it's the protein <span class="Apple-style-span" style="color: #3d85c6;">Bride of Sevenless (BOSS)</span> who's mutant</li>
<li>Boss is a 7pass transmembrane ligand on the R8 cell, and binds to the R7 Sev RTK (see, Sevenless is an RTK, a receptor. BOSS is the ligand. That's why it's "bride of Sevenless," because it binds to the Sevenless receptor)</li>
<li>when they bind, the R7 precursor cell is induced to develop into R7</li>
<li>even if other cells express Sev (which they do), only R7 touches R8, so only the Sev on R7 receives the ligand</li>
<li>Steps to signal transduction</li>
<ol><li>Boss binds Sev-RTK</li>
<li>Sev-RTK is phosphorylated and an adaptor DRK with an SH2 domain docks</li>
<li>a special Ras-GEF called <span class="Apple-style-span" style="color: #3d85c6;">Son of Sevenless (SOS)</span> is also bound by DRK's 2 SH3 domains</li>
<li>SOS makes membrane-bound Ras get a new GTP and the activated Ras continues the signal with cytoplasmic free proteins</li>
<ul><li>see, SOS is Son of Sevenless because it's activated when Bride of Sevenless binds to Sevenless receptor = love child (haha, biologists are both bored and lewd)</li>
</ul></ol></ul><li>Now that Ras is active, where do we go next?</li>
<ul><li><span class="Apple-style-span" style="color: #8e7cc3;">Ras-MAP-kinase signaling pathway</span>: Ras activates MAPKKK (<span class="Apple-style-span" style="background-color: magenta;">Raf</span>), who activates MAPKK (<span class="Apple-style-span" style="background-color: magenta;">Mek</span>), who activates MAPK (<span class="Apple-style-span" style="background-color: magenta;">Erk</span>), who activates gene regulatory proteins in the nucleus</li>
<li>these gene regulatory proteins quickly turn on "early genes," who are part of the <b>primary response</b></li>
<li>early genes produce more gene regulatory proteins that go to activate later genes for the <b>secondary response</b></li>
<li>in this way, there is a rapid response and a more delayed response</li>
<li>this pathway is how the signal goes from surface (where RTKs and Ras await) to the nucleus (where DNA and genes hide)</li>
<li>lots of positive and negative feedback: MAP kinases activate both downstream targets and its own activators, BUT they also increases transcription of phosphatases and deactivate its own activators (Erk blocks Raf)</li>
</ul><li>different sets of 3 MAP kinases form MAP modules in different pathways</li>
<ul><li>however, many modules share one or more of the same kinases</li>
<li>scaffolds prevent cross-talk by associating with the sensor, so that activated kinases that bind to the scaffold are automatically next to its downstream targets also docked to the scaffold protein</li>
<ul><li>this makes sure the activated kinase doesn't wander off and find other targets of other pathways</li>
</ul></ul><li>Rho GTPases regulate the cytoskeleton (actin and microtubules) in response to special guidance receptors</li>
<ul><li>inactive Rho is bound to GDI, which prevents association with the GEF</li>
<li>Eph RTKs are the receptors that activate surface-bound GEFs that activate Rho</li>
<li>ephrins are the guidance ligands that bind Eph RTK</li>
<ul><li>e.g. most commonly used for directing axonal growth</li>
<li>target cells express the appropriate ephrin on its surface</li>
<li>the axon's growth cone has Eph RTK that binds ephrin</li>
<li>Rho changes the cytoskeleton to grow in the direction of the bound ephrin to shift the axon thataway</li>
</ul></ul><li><span class="Apple-style-span" style="color: #a64d79;">PI3-kinase</span> is another protein that docks to RTK</li>
<ul><li>PI3 phosphorylates PIs, usually to make PIP3</li>
<li>PIP3 floats around until the <span class="Apple-style-span" style="color: #a64d79;">PTEN</span> phosphatase turns it into PIP2</li>
<li>PIP2 is nabbed by PLCbeta or PLCgamma and cleaved into IP3 and DAG for signaling</li>
<li>RTKs activate PI3-kinase to make PIP3 available to make PIP2 available for signaling</li>
<li>PIP3 have special PH modular domains (pleckstrin homology) that are super specific (because PIP3 and PI3 tend to be involved in growth and related pathways that are vulnerable to become cancerous)</li>
</ul><li><span class="Apple-style-span" style="color: #cc0000;">PI3-kinase-Akt pathway</span> receives signals from IGF, the "<b>SURVIVE AND GROW</b>" signal</li>
<ul><li>RTK activates PI3 kinase, which makes PIP3, which recruits <span class="Apple-style-span" style="color: #e06666;">Akt</span> and <span class="Apple-style-span" style="color: #e06666;">PDK1 (phosphoinositide-dependent protein kinase 1)</span> to the plasma membrane</li>
<li>Akt is activated there and phosphorylates many targets, producing a growth response</li>
<ul><li>e.g. one target is called <span class="Apple-style-span" style="color: #ea9999;">Bad</span>, which causes apoptosis, but phosphorylation of Bad makes it dock onto a scaffold protein, preventing its function</li>
</ul><li>a special <span class="Apple-style-span" style="color: #e69138;"><b>TOR</b><span class="Apple-style-span" style="color: black;"> protein promotes ribosome production, protein synthesis, and nutrient uptake/metabolism, so it is activated by the Akt pathway for growth purposes</span></span></li>
</ul></ul><br />
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pg. 916 - 935Anuhttp://www.blogger.com/profile/05205515448404134515noreply@blogger.com0