Crazy Postdoc Update

KTVU reports that the Benchum Liu (the accused postdoc) will plead not guilty to poisoning his labmate Mei Cao.  Interestingly, Liu does not deny adding ethidum bromide (EtBr) to Cao’s water (he even participated in a videotaped reenactment for police), but will be arguing that EtBr is not a poison.  My last post talked about EtBr as a carcinogen, and this is certainly what I’ve been taught my whole scientific career. It’s also extremely believable because a) the flat, planar rings make it a great intercalating agent, and b) I know it intercalates because I use EtBr routinely to visualize DNA.  That said, there are some caveats on why it might not be harmful to humans – first off there’s the issue of delivery – merely ingesting a small molecule like EtBr doesn’t guarantee it gets into your cells (in fact propidium iodide, chemically very similar to EtBr, only stains dead cells). Whether EtBr can effect your cells will  be dictated by the ability of EtBr to diffuse across the membrane (or taken up  via protein transporters) and from there it still has to get into the nucleus, all the while remaining soluble.

So what’s known about EtBr in living systems?  Well according to Wikipedia, its used to treat Trypanosomosis (a parasite) in cattle.  That suggests it’s not so bad, as the cattle presumably survive the treatment and (I assume) can be used as food sources for humans.  Well, then the question becomes how does the amount of EtBr that Liu gave to Cao compare to what cattle get?  Rosie Redfield at the RRResearch Blog, writing about common misconceptions about EtBr, did the the following calculation:

“The recommended dose for cattle is 1mg/kg body weight (up to 50mg/kg has been used in mice). Compare this with the 0.25 – 1 microgram/ml used in molecular biology (previous error corrected – thanks, anonymous commenter). A 50kg researcher would need to drink 50 liters of gel-staining solution to get even the non-toxic dose used in cattle.”

For reference, I use about 1 ul (10^-6 L)  of EtBr per experiment (diluted in 50 mL buffer) and have never seen more than 20 mL of EtBr just sitting around (having 50 L of it would be strange to say the least). So does Liu have a case?  I would say that evidence suggests that EtBr WILL cause DNA mutations, but EtBr probably DOESN’T get into living cells at even high concentrations.  What does that mean?  Cao will probably be okay – in fact the National Toxicology Program (NTP) report on EtBr has a puzzling description of it as “Bitter tasting dark red crystals from alcohol (Budavari, 1989)”, suggesting Cao is not the first person to digest EtBr. That still doesn’t change the fact that Liu did expose her to a potential carcinogen that has very stringent disposal guidelines in his lab, and I’m betting his lab is going to get fined for improper disposal at the very least.  It’s also important to note that the evidence in cattle suggests EtBr won’t harm humans, BUT there hasn’t been a carefully controlled study to show that.  Furthermore, there IS evidence that it can act as a mutagen once inside a cell, so given a choice between ingesting it and not ingesting it a reasonable person would choose the latter. I don’t know what this means legally, but it looks like the case has taken yet another unexpected turn, and this time I was able to learn something along the way.

When Postdocs Attack: The UC Poisioning Case and How EtBr Works

We interrupt our scheduled series of posts to bring you the case of the crazy postdoc (for those of you outside the scientific research community, postdoc = postdoctoral researcher, which is the status you attain after receiving your PhD but before anyone wants to pay you a real salary).  The San Francisco Chronicle first reported this case on Nov 12.  The basic summary of the case is that a UCSF postdoc (Benchun Liu) decided, for no apparent reason, to poison his labmate’s (Mei Cao’s) drinking water.  There does not appear to be a motive, but the case is still unfolding and there still seems to be a lot of confusion about the facts.  For instance there’s the mysterious identity of the poison.  Initially the Chronicle quoted UCSF police Capt. Paul Berlin:

“He told investigators that in both attempted poisonings, he had used a buffer agent designed to control acid in lab solutions, Berlin said.

The agent turns water blue, but Cao drank it anyway, Berlin said.”

Well, pretty much anyone with any chemistry or biology lab experience can make an educated guess that the blue pH buffer is most likely bromothymol blue. Bromothymol blue is used in a lot of loading buffers (think those agarose gels with bright blue bands you might see on CSI when someone is running a DNA test) – it’s yellow when pH is low, blue when pH is high.  At first glance bromothymol blue doesn’t seem like the worst thing to digest – after all its use as a pH indicator suggests it’s relatively inert (as in we add it to our solutions because we don’t expect it to do anything but inform us about our solution pH and who cares if your stomach turns yellow if it’s not doing anything bad?).  On the other hand we also use bromothymol blue at very low concentrations (i.e. 0.001%), and pretty much anything is toxic at high concentrations.   We can check the MSDS (material safety data sheets: the papers that contain all the basic information about the chemicals you work with in lab) to see what’s known about bromothymol blue. This MSDS suggests that ingestion of large doses can cause upset stomachs, this one suggests inducing vomiting immediately after any accidental ingestion.  My guess is that no one has really explored what happens when you swallow bromothymol blue because a) it’s a horrible drug candidate so no one’s done toxicity tests and b) no one has ever accidentally swallowed the contents of the very conspicuous, very blue, tube on their bench.  Still all the evidence would lead me to believe that, while it’s not good for you, swallowing bromothymol blue is also not the worst thing in the world.

However, this story took a completely different turn on Nov 25, when the Chronicle reported this:

“Liu is accused of trying to poison co-worker Mei Cao, 44, by putting a laboratory chemical, ethidium bromide, into a cup near her work station.”

Ethidium bromide (EtBr) is vastly different than bromothymol blue.  For one thing, it’s red not blue (although it dissolves colorlessly into large volumes).  It’s also a carcinogen (cancer causing chemical) and teratogen (causes birth defects).  In a lab the most common use of EtBr is to visualize DNA.  Remember those CSI gels?  Well, when you load DNA into them you don’t actually see it running through the gel. You need to put it under a UV light so that it will fluoresce.  But DNA by itself is not easily visualizable so we treat the gels with EtBr and visualize the DNA indirectly through EtBr.   We can do this because EtBr is an intercalating agent – that means it inserts periodically into DNA strands.  That’s great for looking at DNA on a gel.  It kind of sucks when it’s intercalating in the DNA in your cell.  These insertions will cause a lot of DNA damage (breaks, mutations, etc) that can cause cancer or cell death.  In fact when we handle EtBr in the lab we have special disposal for anything that touches it (gloves, pipette tips, gels), because we don’t want it sitting around in landfills.

As an aside and general note of interest, a lot of chemotherapeutic agents are also intercalating agents.  Take a look at the structure of EtBr and a doxorubicin, an anticancer drug:

EtBr

Doxorubicin

See those rings in the middle of both structures?  Those will give the molecules a nice, flat, shape that allow them to slip into a pocket that is inherent in the famous double helix structure of DNA, causing it to unwind.  When dealing with cancer this is an acceptable but risky treatment, because the cancer cells have a greater chance to be effected (since they’re the cells undergoing rapid division and therefore are making lots of DNA).  However, any cell is susceptible to this mechanism of action, so in cases where there’s no cancer, all you’re doing is hurting normal cells and possibly introducing cancer causing mutations (I may give cancer treatments their own post in the future as there’s plenty to say about them, independent of this case).  But, it’s an interesting point that most cancer drugs are also carcinogens.

Anyway, back to the crazy postdoc case: EtBr is considerably worse than bromothymol blue, and looks nothing like it, making me wonder if it’s confusion by the reporter about the science, or if the postdoc is just that crazy and keeps making up different stories (or used both!). In an even weirder twist on the case, the postdoc has been released while awaiting trial – that seems odd to me in an attempted murder case.  Anyway, the whole case is extremely crazy, and we’ll be following it here as updates come.  So far, I think the best line (pointed out to me by a friend) comes from the most recommended comment on the sfgate story:

“It’s the urology department, so his motive is obvious. He was pissed off.”

Update 11/30/08: Liu has pleaded not guilty, claiming EtBr is not a poison.  He might have a case.  See my update post on why EtBr, even with its intercalating properties, is not as bad as you might believe.

Inner Life of the Cell Explained, Part I – Rolling

So for these next series of posts I’m going to attempt to explain what’s happening in the video.  I should warn you I’m not exactly an expert in inflammation, so if you disagree with an explanation please make yourself known in the comments.    I’m going to use time references from this version of the video, and I’ll split the series into four posts corresponding to the four steps the narrator of the explanation video mentioned: Rolling, Adhesion, Activation, and Transendothelial Migration.

First up is Rolling, but before we get to that, let’s talk about what this video is about.  Although parts of the video are showing general features and processes of any cell, the story here focuses on a process known as leukocyte extravasation.  What does that mean?  Well a leukocyte is a white blood cell, essentially the foot soldier of your immune system.   Extravasation  refers to fluid leaking out of a container.  Leukocyte extravasation is literally white blood cells leaking out of your blood vessels so that they can reach damaged tissue.  Essentially your circulatory system is a highway, allowing your white blood cells to travel around your whole body.  Extravasation is analagous to taking an exit, which is necessarry when you need your immune cells to reach damaged tissue.  This whole video is essentially about the mechanics of taking an exit.

While most of the video does talk about taking an exit, first we have to see how this leukocyte car drives along the circulatory system.  This is the rolling step.  Let’s go frame-by-frame:

 

 

0:10-0:14: We’re inside a blood vessel (essentially standing in the middle of the highway).  Note that we’re not actually inside the cell yet.  A bunch of red blood cells are rushing past us at a fast rate, driven by blood flow.  A few white blood cells creep along the side of the vessel.

0:14-0:16: Now we zoom onto one of those white blood cells and look at the interface between it and the endothelial cell that makes up the blood vessel.  Notice all the bumps on the cell surfaces – those will be key in understanding why the cells “stick” to one another.

0:17-0:22:  An even closer look at this interface.  We’ve zoomed in on the surfaces and can now see individual proteins (those bumps we saw before) making contacts. The camera pans back to let us see that the surfaces of both cells are littered with many of these proteins each making contacts.

Here’s what the explanation video has to say about this portion:

While red blood cells are carried away at high velocity by strong blood flow , leukocytes roll slowly on endothelial cells.  P-selectins on endothelial cells interact with PSGL1 a glycoprotein on leukocyte microvillae.  Leukocytes pushed by the blood flow adhere and roll on endothelial cells because existing interactions are broken while new ones are formed. These interactions are possible because the extended extracellular domains of both proteins emerge from the extracellular matrix which covers the surface of both cell types.

The first sentence should be self explanatory as long as you remember white blood cell = leukocyte and blood vessel cell = endothelial cell.  So what exactly is a P-selectin and what’s PSGL1?  They’re both proteins, P-selectin (yellow chain) is found on the surface of the endothelial cell and PSGL1 (purple chain) is on the surface of the leukocyte.  These two proteins have a certain stickiness with each other (think opposite parts of a velcro band) that lets the leukocyte adhere to the vessel wall.  The blood flow then gently pushes the white blood cell along the surface of the vessel causing the PSGL1 molecules to disconnect and then reconnect to downstream P-selectins.  This also provides a possible reason why the red blood cells don’t also stick to the walls.  Assuming they don’t express PSGL1 (something I don’t know for certain but seems likely), they don’t have any stickiness to the blood vessel.

Ok back to the frame-by-frame:

0:23-0:28:  Now we’re looking at an individual cell – we’re probably supposed to be in the white blood cell but the endothelial cell would have the same basic foundation.  That foundation is the cell membrane which is a lipid bilayer.  Lipids are a class of molecules which stack together to form the membrane which is vizualized here as a sea on which things are floating. The rafts floating on this sea are actually called “lipid rafts” (although some biologists don’t believe these things exist, or that there function is overstated) and they’re ferrying around important receptors and such.  Here’s the explanation video:

The outer leaflet of the lipid bilayer is enriched in sphingolipids and phosphatidylcholine.  Sphingolipid-rich rafts raised above the rest of the leaflet recruit specific membrane proteins. Rafts rigidity is caused by the tight packing of cholesterol molecules against the straight sphingolipids hydrocarbon chains.  Outside the rafts, kinks in unsaturated hydrocarbon chains and lower cholesterol concentration result in increased fluidity.

Pretty much what I said above. Sphingolipids and phosphatidylcholine are both types of lipids.  The membrane varies in rigidity and flexibility by the presence of cholesterol (unevenly distributed throughout the membrane) and some of these lipids have hydrocarbon tails that are not saturated (and therefore more flexible).

0:29-0:36: Ok now back to our story which is an inflammation story.  Our endothelial cell is at an inflammation site so it produces a signal to say “Hey there’s inflammation over here”.  That’s visualized by the orange and green structures at the top.  These proteins from the endothelial cell recognize a receptor on the leukocyte (purple thing on the bottom).  Activating the purple receptor triggers a whole set of pathways within the leukocyte to start the inflammation response (so we leave the cell-cell interface and finally go within the leukocyte).  The explanation video says this:

At sites of inflammation, secreted chemokines, bound to heparin sulfate proteoglycan on endothelial cells are presented to leukocyte 7-transmembrane receptors.  The binding stimulates leukocytes and triggers an intracellular cascade of signaling reactions.

The heparin sulfate proteoglycan is just another protein (made by the endothelial cell) that with the the chemokine activates the surface receptor on the leukocyte (the 7 in 7-transmembrane refers to the number of loops in the receptor).  Activating a receptor in this manner triggers a signaling cascade within the leukocyte.  But that’ll be covered in the activation post.  Next up is the adhesion post – we’ve touched on the basics here, but in the next post we’ll look within the cell and see how it’s organized and ultimately how we’re going to have to change that organization to get the leukocyte out of the circulatory system.

Update 5/25/09: Part II is up!

More “Inner Life of the Cell” Explanations

I’ve already posted twice on this widely seen video, but I haven’t really had a chance to go into explaining it in depth.  The makers of the video already provided an explanation video, and Wayne at the Niches blog did some frame-by-frame analysis, but, judging from the google searches to my blog, people are hoping for a more detailed explanation.  I’ve produced a transcript of the explanation video which I’ll take section by section in the next series of posts.  I’ve transcribed this myself so I can’t attest that it’s 100% accurate – if you notice any errors, let me know.  The basic summary is that this video provides a glimpse of one specific cellular process: the activation of a white blood cell in an inflammation response.  The not-so-basic summary will start with the next post.

Update 11/29/08: Part I- Rolling is up

Update 05/25/09: Part II – “Adhesion” & the Organization of the Cell is up.

While red blood cells are carried away at high velocity by strong blood flow , leukocytes roll slowly on endothelial cells.  P-selectins on endothelial cells interact with PSGL1 a glycoprotein on leukocyte microvillae.  Leukocytes pushed by the blood flow adhere and roll on endothelial cells because existing interactions are broken while new ones are formed.

These interactions are possible because the extended extracellular domains of both proteins emerge from the extracellular matrix which covers the surface of both cell types.The outer leaflet of the lipid bilayer is enriched in sphingolipids and phosphatidylcholine.  Sphingolipid-rich rafts raised above the rest of the leaflet recruit specific membrane proteins. Rafts rigidity is caused by the tight packing of cholesterol molecules against the straight sphingolipids hydrocarbon chains.  Outside the rafts, kinks in unsaturated hydrocarbon chains and lower cholesterol concentration result in increased fluidity. At sites of inflammation, secreted chemokines, bound to heparin-sulfate proteoglycan on endothelial cells are presented to leukocyte 7 transmembrane receptors.  The binding stimulates leukocytes and triggers an intracellular cascade of signaling reactions.

The inner leaflet of the bilayer has a very different composition than that of the outer leaflet. While some proteins traverse the membrane, others are either anchored into the inner leaflet by covalently attached fatty acid chains, or are recruited through non covalent interactions with membrane proteins. The membrane bound protein complexes are critical for transmission of signals across the plasma membrane.

Beneath the lipid bilayer, spectrin tetramers arranged into a hexagonal network are anchored by membrane proteins.  This network forms the membrane skeleton that contributes to membrane stability and membrane protein distribution.  The cytoskeleton is comprised of networks of filamentous proteins that are responsible for the special organization of cytosolic components.   Inside microvillae, actin filaments form tight parallel  bundles which are stabilized by cross-linking proteins.  While (deeper?) in the cystol the actin network adopts a gel-like structure, stabilized by a variety of actin binding proteins.  Filaments, capped at their minus ends by a protein complex, grow away from the plasma membrane by the addition of actin monomers to their plus end. The actin network is a very dynamic structure with a continuous directional polymerization and disassembly.  Severing proteins induce kinks in the filament and lead to short fragments that rapidly depolymerize or give rise to new filaments.

The cytoskeleton includes a network of microtubules created by the lateral association of protofilaments formed by the polymerization of tubulin dimers.  While the plus ends of some microtubules extend toward the plasma membrane, proteins stabilize the curved conformation of protofilaments from other microtubules, causing their rapid plus end depolymerization.  Microtubules provide tracks along which membrane bound vesicles travel to and from the plasma membrane.  The directional movement of these cargo vesicles is due to a family of motor proteins linking vesicles and microtubules.   Membrane bound organelles like mitochondria are loosely trapped by the cytoskeleton.  Mitochondria change shape continuously and their orientation is partly dictated by their interaction with microtubules.  All the microtubules originate from the centrosome, a discrete fibrous structure containing two orthogonal centrioles and located near the cell nucleus.

Pores in the nuclear envelope allow the import of particles containing mRNA and proteins into the cytosol. Here free ribosomes translate the mRNA molecules into proteins.  Some of these proteins will reside in the cytosol.  Others will associate with specialized cytosolic proteins and be imported into mitochondria or other organelles.  The synthesis of cell secreted and integral membrane proteins is initiated by free ribosomes which then dock to protein translocators at the surface of the endoplasmic reticulum.  Nascent proteins pass through an aqueous pore in the translocator.  Cell secreted proteins accumulate in the lumen of the endoplasmic reticulum, while integral membrane proteins become embedded in the endoplasmic reticulum membrane.

Proteins are transported from the endoplasmic reticulum to the Golgi apparatus by vesicles traveling along the microtubules.  Protein glycosylation initiated in the endoplasmic reticulum is completed inside the lumen of the Golgi apparatus.  Fully glycoslated proteins are transported from the Golgi apparatus to the plasma membrane.  When a vesicle fuses with the plasma membrane, proteins contained in the vesicle’s lumen are secreted and proteins embedded in the vesicle’s membrane diffuse in the cell membrane.

At sites of inflammation, chemokines secreted by endothelial cells bind to the extracellular domains of G protein coupled membrane receptors.  This binding causes a conformational change in the cytosolic portion of the receptor and the consequent activation of a subunit of the G protein.  The activation of the G protein subunit triggers a cascade of protein activation, which in turn leads to the activation and clustering of integrins inside lipid rafts.  A dramatic conformational change occurs in the extracellular domain of the activated integrins.  This now allows for their interactions with I-cam proteins, displayed at the surface of endothelial cells.  These strong interactions immobilize the rolling leukocyte at the site of inflammation.  Additional signaling events cause a profound reorganization of the cytoskeleton, resulting in the spreading of one edge of the leukocyte.  The leading edge of the leukocyte inserts itself between endothelial cells and the leukocyte migrates through the blood vessel wall into the inflamed tissue.  Rolling, activation, adhesion, and transendothelial migration are the four steps of a process called leukocyte extravasation

Making “The Inner Life of the Cell”

Even while I’ve neglected this blog, my previous post on The Inner Life of the Cell remains quite popular. David Bolinksy, one of the creators of the video, gave a TED talk on making the movie. His talk is not particularly interesting from a scientific perspective, but one thing that he emphasizes that deserves restatement is the view of the cell as a series of micromachines.  A lot of current biologocial research focuses on understanding how proteins, nucleic acids, small molecules, and all the other contents of a cell,  assemble together to form higher order structures that perform complex reactions.  One of the challenges that has arisen in modern biology is taking large scale datasets that we’ve generated (i.e. the human genome, gene expresssion studies, etc), and using them to understand how all the different parts of the cell talk to one another.  This global approach to biology is relatively young, so understanding, visualizing, and analyzing these data is a huge, new challenge. Videos like this one, while perhaps not the most detailed representations of biology, are helpful in communicating this global view of the cell.  Plus it looks pretty cool.