Egad! Why will the girl not sleep?

I'm not a very good sleeper.  Ask my parents.  They'll tell you stories of me at age 3 or so wandering the house in the wee hours of the morning, because I was just awake.  And I recall many nights, even now, where it will take me 2 hours to fall asleep.  And naps?  Unless I'm so sleepy I'm stumbling, forget about it.  In college they called me the "sleep camel" because I would sleep 5-6 hours per night for a few weeks then spend one entire weekend in bed, catching up.  We all have our own sleep patterns that are unique to us, and very few of us fit the average 8-9 hours per night, 10 pm to 6 am kind of pattern.  So it didn't surprise me when my first daughter exhibited similar sleep patterns to me.  After all, she has my nose, my eyebrows, my smile, why not my sleep habits too?  It got me thinking if there was a genetic basis to sleep behaviors.  And guess what, the answer is absolutely! 

Sleep patterns, or more broadly, circadian rhythms, can be thought of as how we live around that 24 hour day/night cycle.  It is well appreciated that circadian rhythms influence nearly every biological process in our bodies: metabolism, cell cycle, tissue repair, digestion, gene transcription, you name it.  So it should come as no surprise that a complex network of genes and proteins dedicated to keeping us in-sync with the sun. A quick search of the Gene Ontology database for "Circadian Rhythm" produced 487 distinct protein products associated with the day/night cycle. 

The genetic basis of sleep patterns are most easily thought of as complex traits: many genes are involved, and the equation is muddled by environment.  For example, our sleep patterns as a society changed drastically with the advent of electricity.  So how can we separate out what component of sleep behavior is genetic and what is environment, or learned behavior?  One easy way is to look at extreme examples where researchers have identified unusual patterns running in families and performed exhaustive searches to identify single genes associated with those behaviors.  While mutations in these genes are exceedingly rare, one can imagine how subtle differences in these genes, or others yet to be discovered, can contribute to a sort of "genetic sleep fingerprint" that is common in a family.

"Early Bird Gene".

The disorder known as familial advanced sleep phase syndrome (FASPS) is a rare, inherited disorder characterized with early rising.  Individuals typically wake at 4 AM, and go to sleep at an early time as well, about 7 PM.  While this disorder is considered non pathogenic, individuals are normal and healthy, it does cause some psychological difficulties as patients feel "out of step" with the rest of society.

In 2005, researchers at UCSF studied a family with three generations of FASPS sufferers, and identified a gene, CKI delta, or hPeriod2, associated with the early sleep, early rise phenotype (Xu et al, 2005). They reported in 2007 that mice carrying two mutated copies of the mutated Period2 gene exhibit similar shifts in sleep patterns (Cy et al, 2007 and Zheng et al, 1999).  The mice had changes to their metabolism, their hormones (estrogen levels were altered), neurotransmitter levels, and alcohol consumption. Talk about broad effects.  

"Short Sleeper Gene"
Another rare reported sleep phenotype reported in families is that of short sleep duration.  These individuals typically sleep only 6.5 hours or so per night, rather than the 8-9 hours more typical of humans.  The trait was mapped to the gene DEC2 (He et al, 2009).  The gene DEC2 codes for a transcription factor, a protein that controls the expression of other genes.  DEC2, and a related gene DEC1, are found to regulate expression of other circadian rhythm genes such as Clock/BMAL1 and PER1 by hooking onto their promoters, little DNA bits upstream of the gene, and turning on expression (Honama et al, 2002).  And, at least in the case of DEC1, gene expression is regulated by light exposure. 

Transgenic mice that have both copies of their mouse-version of DEC2 gene mutated are more active and sleep less than mice with the normal, functional gene.  But mice that have both copies deleted do not exhibit this phenotype, suggesting the mutation seen in these mice (and by extension in humans) is a functional variant, retaining some activity but altered in either specificity or regulation.

"Night Owl Gene"

And rounding out our sleep gene sampling is a gene that when mutated results in people staying up late.  Well, it's not really been shown in people yet, but in mice, mutations in the gene Fbxl3 resulted in the mice having an "overtime" phenotype, where their circadian rhythm was pushed from 24 to about 27 hours (Siepka et al, 2007).  This gene encodes an f-box protein - a protein involved in the ubquitin ligase/proteasomal  degradation pathway. So, Fbxl3 tags proteins to be degraded and recycled.  Work coming from the Pagano lab showed that the circadian rhythm protein Cry2 was ubiquitinated by Fbxl3, and this results in the protein Clock to be reactivated (Busino et al, 2007).  So, Fbxl3 plays a key role in the timekeeping mechanism in a cell, and without it, well, cells, and mice, don't follow the sun.

This is just a teeny, isty-bitsy sampling of some interesting genes involved in keeping us awake in the day and sleeping at night.  I keep these genes, and the 484 more identified in the circadian rhythm pathway, in mind when my 2 1/2 year old decides that 10.5 hours of sleep per day is sufficient for her, and that 5:30 AM is a perfectly reasonable time to wake. She, like me and her dad and everyone else, was born with her own sleep patterns. I do try to coax her into that extra half-hour in the morning, but I know that as long as I provide the right opportunity and conditions for her, she will get the sleep that she needs.  Maybe she's a sleep camel too. 

REFERENCES:

Busino, L., Bassermann, F., Maiolica, A., Lee, C., Nolan, P. M., Godinho, S. I. H., Draetta, G. F., Pagano, M. SCF-Fbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316: 900-904, 2007

He, Y., Jones, C. R., Fujiki, N., Xu, Y., Guo, B., Holder, J. L., Jr., Rossner, M. J., Nishino, S., Fu, Y.-H. The transcriptional repressor DEC2 regulates sleep length in mammals. Science 325: 866-870, 2009.

Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y., Honma, K. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature 419: 841-844, 2002. 

Siepka, S. M., Yoo, S.-H., Park, J., Song, W., Kumar, V., Hu, Y., Lee, C., Takahashi, J. S. Circadian mutant Overtime reveals F-box protein FBXL3 regulation of Cryptochrome and Period gene expression. Cell129: 1011-1023, 2007.

Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptácek LJ, Fu YH .Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome.   Nature. 2005 Mar 31;434(7033):640-4.Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptácek LJ. Modeling of a human circadian mutation yields insights into clock regulation by PER2.

Cell. 2007 Jan 12;128(1):59-70.

Zheng, B., Larkin, D. W., Albrecht, U., Sun, Z. S., Sage, M., Eichele, G., Lee, C. C., Bradley, A. The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400: 169-173, 1999. 

Mysterious, Magical Milk

It's paper time!!! I told you I was leading up to something.  And here it is!
This paper
"Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells" was published in PLOSone April 2009.  It's free, open access so go and read it!  Or if you want, read on and I'll try to take you through it.

So we established that HAMLET is a variation of the alpha*-lactalbumin protein.  HAMLET is formed when alpha-lactalbumin (encoded by the LALBA gene, remember that one?) is partially unfolded, loosing a Ca2+, and hooking around a fatty acid called oleic acid.  This can occur in acidic environments, like say, in the stomach.  Let's recap that: a major protein found in human breast milk changes to a cancer-fighting molecule when it passes through the stomach.  In a clinical study, topical HAMLET killed off skin cancer cells.  In humans with bladder cancer, an infusion of HAMLET caused tumor cells to be shed and tumors shrank. Interesting.

So this team, coming from the Lund University in Sweeden and the A*STAR group from Singapore, wanted to look at how HAMLET might be killing tumor cells. First they showed in cell culture that HAMLET selectively kills off several type of cancer cells but not normal differentiated cells.  Also, alpha-lactalbumin does not kill either type of cell. They also showed through fluorescent-labeled HAMLET that 50x more HAMLET gets inside tumor cells than alpha-lactalbumin.  So, somehow HAMLET is selectively entering and killing tumor cells, but not normal cells.

Next they turned to the proteasome.  They showed in vitro (not in cells, in cell lysate and chemistry experiments) that HAMLET binds to the 20S proteasome.  They also did co-immunoprecipitation and used antibodies specific to HAMLET to pull it out of a cell slurry, and they showed that particles of the 20S proteasome were attached to HAMLET.  Finally they used immunofluorescence in cultured tumor cells to show that HAMLET and the 20S proteasome were located very close to one another.  These data are not water-tight but are suggestive that HAMLET and the proteasome are interacting.

So, HAMLET interacts with the proteasome and also selectively picks off the tumor cells.  What could it be doing?  The group next showed that HAMLET is resistant to proteasomal degradation, by showing it is resistant to the enzymes trypsin and chymotrypsin, and it also is a partial proteasome inhibitor, by showing proteasomes from HAMLET treated tumor cells don't degrade other proteins.  Using mass spectrometry, they see some changes to subunits of the proteasome in HAMLET treated tumor cells: namely, structural subunits, a nuclear localization signal, and some catalytic subunits.  This suggests HAMLET gums up the normal workings of the proteasome.  They also show this on protein immunoblot; proteasome subunits from HAMLET treated tumor cells end up on a different part of the gel, suggesting the size or composition of those subunits is altered. Finally, they show increased fluorescent staining of proteasomes in HAMLET treated tumor cells, again suggesting the trash can is broken.

That's it.

So where does this get us?  It does appear that HAMLET has an effect on the normal workings of the proteasome.  It gums up the trash can, and the authors speculate this creates a toxic effect on the rapidly growing tumor cells.  Normally, gumming up the trash compactors of the cells cause disease, but this is suggesting mucking with the garbage disposal can be beneficial.

Why tumor cells and not normal cells?  Well, they didn't do any of their proteasome studies in normal cells, and they only looked at differentiated normal cells.  Would it have an effect on proliferating normal cells?

And what is this doing coming from breast milk?  It's great that HAMLET has anti-tumor effects, but what is it doing for babies?  Is it just a random by-product or is it somehow offering early protection for our babes in the early years?  There exist a few anecdotal stories out there of women providing breast milk to cancer-striken loved ones, and they loved ones recover.  Just anecdote, or did the conversion of alpha-lactalbumin to HAMLET help these cancer patients?

Related - does it have to be HAMLET from human milk, or does bovine alpha-lactalbumin converted to HAMLET do the same thing?

Well, just thought that was a pretty little study. As a nursing mom and a cancer researcher (on hiatus) it's things like this that fascinate me and make me glad I'm doing what I'm doing.


P.S.  Sorry this one was long-winded and kinda jargon-y.
P.P.S  You can email comments/questions/concerns/suggestions here!




* anyone know how to make the alpha symbol on the Mac using keyboard strokes?

Gene Wednesday: LALBA

Righto!  Here's post number six, and guess what.... six people signed up to follow this little blog. Talk about motivating factors.

Today is the inaugural Gene Wednesday.  I love genes.  I love genetics.  So here's where I'll spotlight one gene.  I promise, all these posts this week, though they seem disjointed, are leading to something.

Today's gene is LALBA. It codes for the protein human alpha-lactalbumin.  It's located on human chromosome 12q13 (the long arm of chromosome 12).  As you can probably guess by the name, it's the principle protein in milk.  But it's oh so much more.  It binds to to another protein, lactose synthase, and makes lactose, the principle sugar found in milk.  LALBA is only expressed in mammary tissue, and is hormonally regulated.  It is highly conserved among mammals, and mice that have both copies of LALBA deleted make a thick milk lacking lactose that does not sustain the pups, showing that LALBA is involved in lactose synthesis and that lactose is necessary for sustaining infants.



Alpha-lactalbumin appears to do much more than make lactose.  It can bind together into a multimeric protein that has apoptotic properties - it can help kill tumor-like cells but not normal cells in culture.   Some data suggest it also has microbicidal activity And recently, a protein dubbed HAMLET (human alpha-lactalbumin made lethal to tumor cells) is alpha-lactalbumin folded in a different way when exposed to acidic environments that, as the somewhat awkward name suggests, appears to specifically kill tumor cells. So one gene, one protein, lots of different functions.  Neat!

Term Tuesday: Proteasome

Today's term is Proteasome.  Never heard of it?  Cool.  Let's get started then.

 Like every good city, someone needs to take out the trash and the proteasome is the one to do it for a cell. The proteasome is the trash compactor and recycling center for proteins in a cell. The proteasome is a big, barrel shaped protein complex in a cell (we're talking eukaryotes here) that degrades and disassembles bad proteins.  By bad proteins, I mean proteins that were made or folded improperly, proteins that no longer are needed, and proteins that have been damaged, say by heat shock.

The proteasome is made up of several protein complexes that arrange themselves, I kid you not, like a kitchen trash can with a pop-up lid.  The details get my head spinning, so I'll probably not do justice to the amazing research behind discovering how it works.  But here it goes.  The main barrel of the proteasome trash can, called the 20S particle, is made of small proteins aggregating together to make rings. There's 7 rings in the barrel, and the outer two contain regulatory elements that can sense what proteins are entering to be degraded. The lid of the trash can, called the 19S regulatory cap, senses the proteins to be degraded and feeds them into the barrel.  It takes energy to destroy a protein and the energy comes in the form of using ATP, the main fuel in the cell.  The energy spent here is in the binding of the bad protein to the 19S and 20S particle.  There's yet another part of the trash can, the 11S particle, and it regulates degradation of short proteins or peptides.

Proteins are generally tagged for destruction by a small chemical modification called ubiquitin.  Attaching onto the amino acid Lysine, the ubiquitin is placed on there by yet another complex of proteins called ubiquitin ligase.  The ubiquitin ligase is the trash collector: it identifies the proteins in the cell that need to be recycled.  You guessed it, there's several proteins associated with ubiquitin ligase, and it's where the specificity comes into play. When a protein is tagged for destruction, it attaches to the 19S (or 11S if it's small) particle and is fed into the 20S particle.  It's here that it gets chewed into small peptide chains, which can be further degraded and recycled into new proteins.  Nice and tidy.

Proteasomal degradation of proteins plays a huge role in the normal control of cell division, homeostasis, and stress response.  However, sometimes the proteasome degradation process goes awry, and can lead to disease.  Too much proteasomal activity can lead, supposedly, to autoimmune disease.  Not enough can lead to things like Alzheimer's disease and cancer.

Remembering back to high school biology (quite a few years back, I won't tell you how many) I never learned about proteasomes. It's because they probably weren't identified yet as parts of the cell. They were only really discovered in the '80's.  And work that identified it was deemed worthy of the Nobel Prize in 2004.  Really, we all should celebrate those who know how to take out the trash.

P.S.  I'll get to actually putting in reference and attributions here soon.  I promise.

Method Monday: Cell Culture

I have my training in genetics, and more specifically cancer genetics.  So a great deal of my work has been with little cells growing rapidly on plastic, in pink-colored media, in a warm, humid incubator.  This is the world of cell culture, also called tissue culture. It's also the iconic image of a laboratory worker: someone in a white coat, gloves and goggles, laboring away with their arms in a plastic chamber and transferring reddish liquid from one flask to another.

Hint: most of those pictures are totally staged.  I never wore goggles.

Here's what cell culture is all about.

Cells are harvested from a source, usually a hunk of tissue, (we'll come back to that) and are gently teased apart both mechanically via scissors and chemically with enzymes, typically trypsin, collagenase, or dispase, that chew up the connections between the cells.  Then, the cells are soaked in a liquid media, and placed in a vessel, either a flask or a petri dish.  These are usually plastic treated with poly-L-Lysine, creating a positively charged surface to make the cells stick better.  Then the cells sit in an incubator set to body temperature (37C, or 98.6F) that is infused with carbon dioxide.  This keeps the cells at a happy pH. Then let the experiments begin!

What is in the media?  Well, it's got some salts, some sugar, some minerals.  Think of it as a really expensive form of Gatoraide.  Usually there's antibiotics too, because good cells and bad bacteria don't mix.  That can be it, and that is called minimal media - just enough to keep the cells alive.  But most cells need more than that to grow.  So extras are added, such as amino acids, the building blocks of proteins, and often serum.  Harvested from calves or fetal calves (don't ask, you don't want to know), serum a blood product chock-full of growth factors and goodies that help cells grow.  Oh, and the pink color?  It's phenol red, a dye that indicates the pH.  Cherry red is good, purple is bad.

So where do these cells come from? Pretty much any hunk of tissue will do, from human, mouse, dog, Chinese hamster, you name it.  For a lot of purposes, cell lines have already made, and can be purchased.  Very convenient, because it allows multiple researchers to use the same cells in their experiments and it's easier to compare data.  For example, one frequently used cell line is a human cervical cancer cell line called HeLa.  The name comes from "Henrietta Lacks."  The cells were made in 1951, and nearly every cancer lab since has used those cells. Hows that for immortality? Other times researchers have to make their own cell lines.

Some main categories of cells people use are primary, immortal, and stem cell lines.  Primary means they're straight from a living being, and typically have a short time they can be used due to senescence - a process of aging that limits the number of times a cell can divide. In one lab, I routinely made primary cell culture from mouse hippocampal neurons. Not cells that can easily live and divide for many generations.  Immortal cell lines have supposedly limitless replicative potential are those that have been pushed through senescence, through crisis, and live on. Or they have been induced to live on and on by introducing viral genes that allow them to keep on growing.  Both methods rely on changing the expression of genes that normally keep cells from dividing out of control.  And stem cell culture takes cells from the inner cell mass of an embryo which are then grown on a feeder layer of cells, with careful media conditions.  These cells are undifferentiated and supposedly have limitless replicative potential.

So, there's books and books written on cell culture, and really neat things researchers use it for.  I remember in one lab working on muscle differentiation, and I could take muscle stem cells, add a couple growth factors into it and a few days later, watch the differentiated muscle cells twitch in the dish.  Freaky. But other than playing around, cell culture gives us a way to poke and prod into the biology of cells in a way not otherwise possible in a living organism. Yes, there's drawbacks and limitations.  Cells in a dish do not behave like cells living in a body.  But for a vast amount of basic biology and early drug testing, cell culture is a key way to gain insights and knowledge.

Term Tuesday: SNP

Wow.  This little blog has 2 followers.  That's more followers than there are posts.  Crazy.  OK, well, not to disappoint my fans, I will renew my effort to explore the world of biological science, particularly genetics with you.

So... introducing Term Tuesday, where I'll pick some cool term at random and try to shed light on what it means.

Today's term is SNP.  Most people pronounce it "snip".  It stands for  Single Nucleotide Polymorphism.  Kind of a mouthful, no?  Maybe, but it's pretty simple. While most of our genes are made up of the same combination of the nucleotides A, G, C, and T, from time to time there will be one "letter" different.  A single nucleotide. And this small difference is not enough to change the resulting protein product, or whatever that piece of DNA encodes.  It's just enough to be a variation, or polymorphism. Like a different way of spelling the same word.

So why do people care about SNPs?  Turns out they can be very useful.  One example of how they are cool is that SNPs can be used to identify candidate disease genes.  Since everyone carries around a different combination of SNPs, geneticists can compare SNPs in people with a disorder to people without a disorder.  If enough SNPs associate with a disease, there's a good chance a gene associated with the disease is located on the DNA near the SNP.  So SNPs can act as a road mile marker.  They're so useful in fact that researchers have identified common SNPs in the population and put them all on a gene chip, to make the identification process go that much faster.  Once a set of SNPs have been identified, a small chip containing disease-related SNPs can be made, to quickly and economically assess someone's risk for carrying a yet-to-be-identified disease gene or genes.  Neat, huh?

I bet there's lots more about SNPs but that's all I have time for today.   Any driving questions?  Just post a comment!

Shhhhh - quieting the troublesome genes

Welcome to the inaugural post of Why You Will Love Science.   Today we discuss a paper published in the Proceedings of the National Academy of Sciences discussing some really cool emerging technology.  The title, "Lipid-like materials for low-dose, in vivo gene silencing" is a great summary.   This seemed like an excellent paper to start with because it delves into some relatively recently understood fundamental basics of biology, the authors work diligently to show it works across different species, including nonhuman primates, and the work shows some exciting progress towards new types of medicines - specifically in the hot arena of gene therapy. So let's begin, shall we?

So you're not left hanging, here's the punchline.  The authors have developed a way to get small nucleic acid particles targeting specific genes to turn down the expression of those genes into liver cells of animals.  Not only did they turn down the expression - or silence - one gene, but up to five genes simultaneously in the same animal.   And they were able to do this at very low levels of "drug" with no obvious side effects.  Pretty cool? Definitely.

First a bit of background.
siRNA are little pieces of genetic material that takes the product from a gene, the messenger RNA, and diverts it from making a protein, and generally targets it for destruction.  It's a way your cells have of regulating genes, amazingly only discovered about 20 years ago.  This paper looks at using siRNA to do gene therapy - modifying a gene to treat a disease.  This is a tricky thing to do because not only is it really hard to get genetic material into a cell - there's a little something called an immune system in the way - and once genetic material gets to the target cell, it's difficult to generate a sustained response in the cell.

So what did they do?

They first created a library of fat-like particles called lipidoids of varying sizes in a way that was fast (3 days) and could be completely automated.  This is important for future applications.  Next, they tested how effective each little fat bubble was at getting the siRNA into a cell.  Most were not effective but a few looked really promising.  They chose a few really potent ones and tested in mice.

The first test in mice was to target a liver-specific gene, Factor VII.  They chose one particle type that was effective at a very low dose, and added a few modifications to make it more invisible to the immune system and less sticky with other blood proteins, giving the particles an extra chance to get into cells. The most potent one was several hundred fold more potent than the current method.  They dubbed the particle C12-200.  With a very low dose they observed lowered levels of Factor VII for up to 25 days, with little side effects in the mice.

They next wanted to see if they could target multiple genes at the same time.  They added in siRNAs that targeted four other liver-specific genes  At low doses that were well tolerated by mice they could  successfully knock down all five of the genes simultaneously.  Very cool.

Next they looked to see if the best lipidoid would work in non-human primates.  Again they chose a liver-specific gene.  And it worked.  At very low doses.  With no apparent side-effects.

So why are these little fat bubbles so interesting?  Some diseases, such as hepatitis C, have no effective treatments but has unique genes that would be great siRNA targets. We might be able to use these little fat bubbles to target the genes in HepC, shutting them off, and providing a cure.  Or, if you had a disease with multiple genes involved, such as cancer or metabolic syndrome, you could target those genes, altering their expression and stopping the disease in their tracks.

What is still missing?  Well, they only looked at genes active in one organ, the liver.  This made for clean experiments but for other applications, it will be important to know how wide-spread the siRNAs have their effect.  For example, in a cancer cell, turning off a gene might be a good thing, but in a normal cell it might have dire consequences.  This still needs to be tested.  Also, in mice the length of time of gene silencing was quite a long time, but it would be nice to know if they can give a second dose and quiet those genes again.