Alliums: giving the immune system a smelly leg up

By February I am generally fed up with the cold and flu season, and this year is no exception.  Our household has been hit hard by a cold/bronchitis 1-2 punch, and we’ve been single-handedly keeping the makers of Ricola and our local Pho (Vietnamese chicken soup) shop financially solvent for the last few weeks.

It’s about this time that I start musing about zinc, antivirals, ICAM-1 inhibitors, VP4 protein based vaccines…in short, why the heck haven’t we found a way to beat the common cold?  Continue reading


Bitter but beneficial: salicylic acid and willow bark

After the last half-marathon I ran two weeks ago my longer runs have been hampered a bit by a nagging inflammation in my right lower hip muscle (piriformis strain: it’s a pain in the butt).  I was reflecting on this mild misfortune this weekend as I was reaching the home stretch of a very pretty 12-miler near the water, which went right past a clump of willows in early flower.  (Salix lasiolepsis…I think.  Salix laevigata is also native here, but the loose open buds looked more like lasiolepsis).  This provided an ample reminder of the general awesomeness of willow bark’s key pharmaceutical component, salicylic acid, and its more famous derivative, acetylsalicylic acid.

Willows by the water: a stand of flowering Salix.

Willow flowers just past the fuzzy pussy-willow stage. These look right for S. lasiolepsis; S. laevigata flowers stay more compact and fuzzy.

Good for a whole range of what ails you:

One of the more well-publicized botanical myths is that aspirin comes from willow bark.  This is only a short hop from the truth.  The actual anti-inflammatory produced metabolically by willow is salicylic acid–depending on the species and time of year, willow bark contains between 0.08 – 12.6% salicin, the metabolic precursor of salicylic acid.  Salicylic acid (historically purified in large quantities by boiling the bark of white willow, Salix alba) is readily acetylated into acetylsalicylic acid, trademarked as Aspirin.  You might have made acetylsalicylic acid back in chemistry class, or you leave aspirin tablets somewhere damp, you might notice a vinegary smell as they hydrolyze back to salicylic acid and acetic acid. Curiously, while Bayer still holds the trademark on “Aspirin”, “aspirin” is generic.  A/aspirin (hmm…not sure what the trademark rule is on capitalization at the start of a sentence) is part of the NSAID group of anti-inflammatory drugs–useful at reducing fever and throbbing pain.   Aspirin also exerts a potent anticoagulant effect by inhibiting the platelet aggregant thromboxane, so many people take it to reduce the risk of stroke or blood clots following surgery.

Salicin and salicylic acid, found in willow bark, and acetylsalicylic acid, better known as aspirin.

Salicyclic acid is also anti-inflammatory but is probably more recognized for its antibiotic effects; it’s one of the most common topical treatments for the bacteria that cause acne.  This handy dermatological property is of course just a human co-option of the plant’s own defense system: salicylic acid is produced by plants in response to stress, and reduce their vulnerability to bacterial infection.

It’s not all good news though…

The reason we don’t take salicylic acid orally as a pain killer is partly because the pharmacokinetics are a little different from aspirin, but mostly because salicylic acid is very rough on the stomach.  Acetylsalicylic acid is less irritating (for reasons I have been hard-pressed to discern–it’s a weaker acid, but at stomach acid pH that shouldn’t make a difference), but it can still cause ulcers and stomach bleeding if taken in too high an amount or with other NSAIDs.  What’s more, the conjugate base of salicylic acid, salicylate, is damaging to the hair cells of the inner ear in some people (salicylate sensitivity).  Chronic aspirin consumption can cause tinnitus when the acetylsalicylic acid hydrolyzes back to salicylic acid in the bloodstream.

Extra nitty gritty: this bitter pill to swallow has a pleasant aftertaste

So. Pretty versatile, right?  A quick hunt through the scientific literature for salicylic acid entries yields a complex mixture of rheumatology, dermatology, cardiology, botany, auditory and gustatory references.

Wait, gustatory…?  Yes.  Because as you know if you’ve ever had the misfortune of having to down an aspirin without a glass of water, salicylic acid and acetylsalicylic acid taste just awful–they’re the very definition of bitter.  This was, in fact, a significant hitch in marketing acetylsalicylic acid early in its commercial development, and pharmacologists tried out a range of less effective and even toxic variants to try to make it more palatable before resigning themselves to just mixing it into a sugary syrup or coating the pills.

Curiously, the bitterness of salicylic acid and its derivatives has become an independent asset to science, because they’re the perfect compounds to study the physiology and chemistry underlying bitter taste detection.  Salicin (basically salicylic acid with a glucose ether instead of a carboxylic acid) was recently used to study the crystal structure of the “bitterness” taste receptor hTAS2R16.

Why aspirin tastes so bad: two proposed models for the interaction of salicin (blue) with key amino acids (green) in the bitterness taste receptor hTAS2R16. From Sakurai et al., J. Biol Chem 2010.

So if you find yourself near fresh water in the next couple weeks, enjoy the budding willows, but don’t take a bite out of the bark.


Prettiest Plant in the Lab: Foxgloves, Digoxin, and Digoxigenin

A while ago I posted about oleander and the structural similarities between the oleander cardiac glycoside oleandrin and the foxglove cardiac glycoside digoxin.   But foxgloves express enough biologically useful (and harmful) molecules that they’re worth showcasing.  Plus they’re such nice eye-candy!

Gloves that pack a punch

The various members of the Digitalis genus, which include the gardener’s favorite foxglove Digitalis purpurea and a range of other Digitalis species, are favored ornamental plants for their tall showy flower spikes and bright colors.  A disputed but appealing origin for the name was advanced by William Henry Fox Talbot, who proposed that the whimsical ye Olde English people imagined fairies wearing the deep cone-shaped flowers for gloves, and called them folks’ gloves.  Very cute.  But given that fairies were famous not only for being adorable but also for other light-hearted mischiefs like stealing babies and poisoning livestock, it’s perhaps fitting that their pleasing-to-look-at gloves come barbed with a heavy dose of serious poison.

Enough folk's gloves for a children's book full of fairies. Digitalis purpurea, by Ferdinand Bauer.

Foxgloves are poisonous because they contain two cardiac glycosides, digoxin and digitoxin, which are found in all parts of the foxglove plant but are most concentrated in the leaves.  There are a few ways to poison yourself with foxgloves by mistake: the flowers have some appealing similarities to honeysuckle, which might lead the unwary to try to suck nectar from them.  Because the plant is still poisonous when dry, a hapless gardener might inadvertently inhale foxglove plant matter when digging or replanting near an old foxglove bed.  The leaves of foxglove (especially before it flowers) resemble and have sometimes been mistaken for comfrey, which is benign and a common basis for tea (here’s an article about several people poisoned this way).  Never fear though, ehow has a handy how-to on distinguishing comfrey from foxglove leaves; the clearest difference might be that foxglove leaves are finely toothed while comfrey’s are smooth.

Leaf of comfrey, Symphytum sps. Good for tea, beloved by herbalists, and pretty easy to confuse with foxglove leaves, pictured in the illustration above. Photo courtesy of Heather at, which is also a pretty nifty blog.

Finally, one of the side effects of digitalis poisoning is strong hallucinations, so there may be a handful of people out there ingesting it deliberately…but I doubt the visuals are worth the heart arrhythmias, severe nausea, fainting, coma and possible death that come with them.

Extra nitty-gritty: Digoxin as heart medicine

Cardiac glycosides like digoxin and oleandrin work as sodium-potassium ATPase inhibitors, which means that they interfere with the balance of ions inside cells. The muscle cells of the heart are particularly vulnerable to changes in sodium concentration, because sodium concentration is coupled to calcium export, and the calcium concentration inside the muscle cell is what regulates how strongly or quickly the muscle cell can contract.  When there’s too much sodium, the cell can’t efficiently export calcium, and the cell contracts too strongly as a result.  Erratic muscle contractions are certainly bad news for a healthy heart.

Digoxin: sometimes a help and sometimes a hindrance to heart function.

Unlike oleandrin however, digoxin has considerable utility as a medicine: the same calcium ion hoarding effect that’s so dangerous in a healthy person can be used to combat heart failure by promoting stronger contractions in a damaged heart, and digoxin gained FDA approval as a treatment for chronic heart failure and some kinds of heart arrythmias in 1998.

The initial use of digoxin came before beta-blockers were used to manage heart failure (HF), and there is ongoing study as to whether digoxin remains valuable as an HF management strategy in concert with other therapies. A recent article in the International Journal of Cardiology has undertaken a multivariable regression approach to attempt to classify which categories of patients are more likely to suffer higher mortality or further hospitalizations for heart failure following digoxin use.  Their meta-study combined cases of over 7000 patients, and found that higher mortality and hospitalizations for heart failure were correlated with groups of patients that were female and had high blood pressure.  Studies like this one may help identify which groups of patients can still benefit from digoxin and which groups should avoid it.

Extra nitty-gritty II: Digoxigenin as molecular label

Apart from its cardiac glycosides, Digitalis also harbors a supremely handy steroid, Digoxigenin (DIG), which I use routinely to label RNA molecules.  Digoxigenin is a fairly small little molecule that can be coupled to the nucleotides that make up DNA or RNA (nucleotides=letters: A,G,T/U, and C), and there are specific antibodies for DIG that can be used to detect it anywhere it’s bound in a cell.

DIG-UTP. This labeled "U" is incorporated into RNA molecules just like regular UTP.

So when I want to find which cells in my tissue sample are making a certain RNA, I can make a probe with a complementary sequence and some of the U’s labeled with DIG.  Then I can use anti-DIG antibodies (conjugated to an enzyme that makes a purple color under the right conditions) to look for the probe, with a technique called in situ hybridization.

A little how-to for using DIG-labeled UTP to find a target RNA by in situ hybridization.

Want more details?  Here are references for the articles mentioned:

Ather S, Peterson LE, Divakaran VG, Deswal A, Ramasubbu K, Giorgberidze I, Blaustein A, Wehrens XH, Mann DL, & Bozkurt B (2011). Digoxin treatment in heart failure – unveiling risk by cluster analysis of DIG data. International journal of cardiology, 150 (3), 264-9 PMID: 20471706
Lin, C., Yang, C., Phua, D., Deng, J., & Lu, L. (2010). An Outbreak of Foxglove Leaf Poisoning Journal of the Chinese Medical Association, 73 (2), 97-100 DOI: 10.1016/S1726-4901(10)70009-5

Poppies part deux: taxonomical embarrassment

Botanical illustrators go crazy for poppies, so when I was looking for pictures to put in Monday’s post, there was one particular botanical illustration of Papaver somniferum I was looking for–one I’ve seen in maybe a dozen places, so I thought it must be the iconic, quintessential opium poppy illustration.  And I spent like half an hour on Google images, digging through variations on “Papaver somniferum red,” Papaver somniferum botanical,” Papaver somniferum painting…drawing…red…poppy…opium poppy red…opium poppy drawing….aaaarrggghhh!  Couldn’t find it anywhere.

But! I knew I had it in one of my botanical books at home.  So once I got back, I pulled out my copy of Wilfrid Blunt’s “The Illustrated Herbal,” found “poppy” in the index, turned the page, and cried out to JMG: “Ha!  See!  It’s right here, by Rinio!  Why was this not findable in Google?? Everybody loves this painting, it’s like the perfect painting of an…oh.  OOOOOhhhh.  It’s a corn poppy.  Well, dammit.”

Rinio's "Papaver rhoeas," the corn poppy. Not an opium poppy, it turns out.

Corn poppies are the ones from the poem “Flander’s fields” (the WWI poem you may have been forced to memorize in high school).  They grow wild in much of Europe.  And while the deep evolutionary conservation of alkaloid biosynthesis machinery in the Papaveraceae means they probably make a bit of the opiates their sibling species is known for, it doesn’t count as an opium poppy.  Hrmph.

In my defense, they look awfully darn similar.  As far as I can tell the main differences are the anther distribution and color in the center of the flower, and the size and roundedness of the seed capsule.  The foliage looks more feathery in the corn poppy too, but I think this varies among subtypes of the two species.

Why I am not a taxonomist: Opium poppies, Papaver somniferum, var "cherry glow" for purchase from Capital Gardens in the UK.

Poppies: You’re getting sleepy…very sleepy…

No you’re not, wake up! You haven’t even started reading yet!

I suffered a nasty shock recently when I discovered that the audaciously sunny California poppy is called “Eschscholzia californica.” Eschscholzia? Bleh. Such a slushy mouthful to hang on a cute little flower. But despite the disservice done to it by Linnaeus, the California poppy has held up bravely, bringing sunshine and some surprising biochemical utility to our coast.

Undaunted by its miserable Latin name, a California poppy brightens up the median.

Doped up on poppy seeds

Ever since I watched the Mythbusters episode where Adam and Jaime were able to make themselves test positive for morphine and codeine by gobbling poppy seed cake and bagels, I’ve been curious about how widespread the expression of narcotics in related flowers actually is. The first surprise was that the poppy seeds we eat are straight from Papaver somniferum, the same opium poppy used to make, well, opium. Can someone with FDA expertise please explain how possession of poppy seeds is no problem, and growing opium poppies in your yard is totally cool (really, some of my neighbors have them right out front), but you could never do that for cannabis or coca plants?

Meanwhile, both California poppies (genus Eschscholzia) and opium poppies (genus Papaver) are members of the Papaveraceae family, which has been around for 70 million years or so, and these two genera are roughly 30-50 million years distant from each other in evolutionary time. So what is the likelihood that the biosynthetic machinery for morphine and codeine and the like is shared between them? Surprise #2: quite high!

Morphine and codeine are both alkaloids made via several steps from the amino acid tyrosine. California poppies have all the enzymes involved in this pathway, but because of the bias in the way those enzymes are used, very little of these narcotics are made in California poppies, with several other alkaloids being favored instead. (Everybody got that?  So don’t go off and start smoking CA poppies now, ok?) One of the benzophenanthridine alkaloids made by California poppies, sanguinarine (named for the bloodroot where it was first found), is worth a little more discussion.

Do I have any poppy seeds stuck in my teeth?

In the 1990s, sanguinarine was found to be a potent antimicrobial, and so effective against plaque bacteria that the company Viadent added it to their toothpaste and mouthwash for several years…until it was found that it also killed off human cells, leading to precancerous mouth lesions. Yuck.

mmm...toothsome sanguinarine

But wait! That actually was handy for sanguinarine, because in the course of characterizing its toxicity to human cells, it was found to preferentially target dividing cells, and where are dividing cells a problem? In cancer of course! Consequently, sanguinarine and a related E. californica alkaloid, chelerythrine, are under investigation for their antiproliferative and pro-apoptotic effects in prostate cancer.

Extra nity-gritty: Poppies in the lab

At the same time, the actual yield of any of these compounds from living poppy plants is pretty low, so a fairly rigorous side discipline has sprung up around trying to get E. californica to make more of sanguinarine and other useful alkaloids. One approach has been to find the rate-limiting enzymes of benzophenanthridine alkaloids and force the plant to express more of them. Another has taken advantage of the fact that all these alkaloids are made as a stress response in plants, and has involved stressing the plant with everything from aspirin to yeast extract in order to drive up their production.  Both approaches use a cell culture system based on small chunks of E. californica tissue, and I derive a small amount of joy from imagining racks of agar petri dishes filled with poppy plantlets.

Want more detail? Here are references!

Adhami VM, Aziz MH, Reagan-Shaw SR, Nihal M, Mukhtar H, & Ahmad N (2004). Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery. Molecular cancer therapeutics, 3 (8), 933-40 PMID: 15299076

Cho, H., Son, S., Rhee, H., Yoon, S., Lee-Parsons, C., & Park, J. (2008). Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures Journal of Biotechnology, 135 (1), 117-122 DOI: 10.1016/j.jbiotec.2008.02.020

Takemura T,, Chow YL,, Todokoro T,, Okamoto T,, & Sato F (2010). Over-expression of rate-limiting enzymes to improve alkaloid productivity Methods Mol Biol, 643, 95-109 DOI: 10.1007/978-1-60761-723-5_7

Oleander: backyard killer has a softer side?

A murderer lurks in your neighborhood.  It’s Nerium oleander, and it’s everywhere. Great swaths of it envelop LA freeways.  It’s littered across backyards, and encircles parking lots.  And it’s deadly poisonous to humans, animals and especially kids so don’t taste it, don’t sniff it, don’t even touch it, OMG I brushed against it AAAHHHH!!!

Don't be fooled by the sweet pink exterior. It's totally out to get you. (Photo by Servophbabu, Creative commons attribution 3.0 unported license)

Actually, it may not be quite that big a deal.  Although all parts of the oleander plant are toxic, and quite a lot of people are treated for oleander poisoning in the US, there have actually been only a handful of adult deaths from oleander poisoning in the last 25 years.  Most of these were deliberate self-poisonings, with the exception of a young couple of vegans who got lost while hiking and ate a whole mess of oleander leaves.  Estimates I’ve seen suggest that a lethal dose to a child would be about one whole leaf, and several leaves for an adult, and the potential for poisoning by skin contact is minimal.  So, while you should definitely watch out for your kids around oleander, you don’t have to be quite as afraid of it as I always thought. The urban legend about a troop of boy scouts who died after roasting their marshmallows on oleander sticks, for example, is almost certainly bunk.

That having been said, getting sick from incidental leaf consumption would be no fun, and oleander really is everywhere, so here’s what to look for:  it’s a tall shrub, 2-6m high, often used in neighborhood hedges.  If you live anywhere in southern California (or some places in northern California, like along the 80 between Davis and Sacramento), you’ve seen it lining the freeways and medians.

A menacing stretch of median oleanders. (Photo borrowed from, but original photographer unknown).

The flowers are vibrantly pink or red or sometimes white, and grow in bunches.  Most distinctive are the leaves, which are dark green and leathery, and shaped like thin daggers about 4-8 inches long (there’s that assassin imagery again).  The leaves and flowers are poisonous because of several compounds, but notably the cardiac glycoside oleandrin/oleandrine and its metabolites.  Cardiac glycosides interfere with the Na+/K+ ATPase pump in heart muscle cells, throwing off the balance of ions inside the cell, and ultimately leading to it contracting faster and more strongly than it’s supposed to.  So oleander poisoning can result in irregular heartbeat, poor circulation, seizures, coma, and death.

Oleandrin. My heart's all a-flutter just looking at it.

Myth busting

While I was in college near LA, I encountered a rumor that oleanders were heavily planted along the local freeways because they were able to metabolize carbon monoxide (CO) emissions from cars, thus cleaning the air.  Turns out, the rumor may have had it exactly backwards.  A whole litany of plants, maybe all of them, can take up and metabolize small amounts of CO from the air—this was determined by Canadian botanists Bidwell and Fraser in the 1970s, who put radioactive C14-labeled CO into the air around plant samples, and recovered the C14 label from the plant later.  But the only study that talks specifically about oleander and CO is by Fischer and Luttge in 1978, who found that oleander was actually a net producer of CO through C1 metabolism of glucose.  That is to say, they might take up a little CO from the air, but then they actually make more of it in their own metabolism.  So no air-quality help there.

Extra Nitty-Gritty: Plant crossover!

One of the treatments for oleander poisoning is a digoxin immune fab, using antibodies raised against a similar glycoside from the plant foxglove called digoxin.  Apparently, oleandrin and digoxin are similar enough that antibodies raised to the latter will also bind the former.  The antibodies work by binding to the glycoside and preventing it from reaching its target in the body and causing harm–it’s the same principle used to make snake or spider antivenom.

Oleandrin and digoxin. You can see how the pink-highlighted parts are almost identical, so that some antibodies that are made to bind to digoxin will also bind the corresponding parts of oleandrin.

Anything oleander is good for?

In previous centuries, oleander was used as an herbal medicine to treat everything from headaches to eczema, which makes me queasy to think about.  Since digoxin is used therapeutically for some heart conditions, I thought oleandrin might be the same, but its therapeutic index seems pretty limited in that context.  However, it does appear that the same Na+/K+ ATPase pump interfering properties that make oleandrin so dangerous to heart cells also make it effective at killing off some kinds of cancer cells when used in concert with chemo- or radiotherapies.  But a drug based on oleandrin called Anvirzel stalled after Phase I clinical trials in Ireland (due to poor performance–no reduction in solid tumors was seen but side effects were, and at least one company trying to inflate claims of its efficacy and continue to sell it got in serious trouble with the FDA). Now it’s the subject of an alarming cancer home-remedy fad based on making oleander extract at home.  People, please don’t poison yourselves!

Want more detail?  Here are the references:

Fab antibody fragments: some applications in clinical toxicology.  Flanagan RJ, Jones AL. Drug Saf. 2004;27(14):1115-33.

Cardiac glycosides in cancer research and cancer therapy. Winnicka K, Bielawski K, Bielawska A. Acta Pol Pharm. 2006 Mar-Apr;63(2):109-15.

Phase 1 trial of Anvirzel in patients with refractory solid tumors.  Mekhail T, Kaur H, Ganapathi R, Budd GT, Elson P, Bukowski RM. Invest New Drugs. 2006 Sep;24(5):423-7.

Cyclopamine, it’s just what it sounds like

Allow me to paint you a picture:

You’re walking through a meadow in Idaho on a lovely warm day.  The grass waves softly in the breeze, and your picnic basket swings loosely from your hand.  The rolling hills are dotted with sheep and flowers.  You turn a corner, and walk into this:

Nooooooo!!! Get away from my picnic basket!. (Public domain image from the USDA).

GAAAAHHHH!!!! You fling your picnic basket at it, spattering wine all over yourself in the process.  What was that??

It’s a one-eyed sheep of course, and it got that way because its mother ate this plant:

Lovely, no? But deadleah. (Photo by Jerry Friedman 2008, used under the Creative commons attribution-Share alike v3.0 uported license)

Which is the corn lily Veratrum californicum (it grows in the Sierra Nevada and Rocky mountains, but the one-eyed lambs that understandably freaked everyone out were in Idaho).  Also known as false hellbore or cow cabbage, it’s a tall plant, about 4-6 feet high, and the leaves, stems, and especially roots produce a robust amount of this compound,  cyclopamine:

It looks harmless enough.

…so named because…well, yeah.  It was causing the farm animals that ate it to give birth to cyclopic babies.

How does it do that?

A better question is: how do you normally end up with two well-positioned eyes, anyway?  There has to be a signal in early embryonic development to tell your facial organs where and how many to form.  An easy way to do this is to tell cells how close they are to the middle of the face.  It turns out that a major “middleness” signal is a protein called Sonic hedgehog that comes from a structure in the developing brain. (Or depending on context, just Hedgehog.  I swear.  Maybe later I’ll write a post on how genes and proteins get such crazy names).  All the cells nearby have another protein, Smoothened, that relays how strong the Hedgehog signal is to the inside of the cell.  If the signal seems strong to the cell, it knows it’s in the middle of the face (nose territory, for example), and if it seems weaker, it knows it’s off to one side.  But when cyclopamine is around, it interferes with Smoothened, so the cells can’t “hear” the Hedgehog signal. Which means not enough middle-of-the-face forms, and structures that should to be further apart think they ought to form right next to each other in the middle.

Losing Smoothened's activity because of Cyclopamine is a bit like taking a picture of a face, cutting down the dotted lines, and gluing the two halves back together. Without enough middle, the eyes end up too close together, or even fused.

So, voila: the cyclopamine in the lily is eaten by the mother sheep during pregnancy.  It gets in the way of Hedgehog doing its job in the embryo, and the eyes creep too close together, in some cases fusing into just one eye.

Can this happen in people?

Yes.  While humans aren’t likely to be exposed to cyclopamine during pregnancy, there are mutations in the gene that encodes Shh or its partners that can cause exactly the same effect (in humans, the resulting condition is termed holoprosencephaly).

Is that what happened to Leela?

Bus driver: "nice eyeball, eyeball." Leela: "nice ass, ass."

Probably not, because Hedgehog signaling is also used for lots of other things, including making fingers and functional kidneys, which seem just fine on Leela.

So that seems pretty awful.  Does it do anything useful?

As it happens, Hedgehog signaling is crucial for the progression of some cancers, including basal cell carcinoma and medullublastoma.  Blocking Hh signaling through the use of Cyclopamine (either as a direct extract from V. californicum or through its derivative IPI-926), is being tested in treatment of these and other cancers.

Want more details?  Here’s a useful review of Hh, cyclopamine, and cancer:

Targeting the hedgehog pathway: the development of cyclopamine and the development of anti-cancer drugs targeting the hedgehog pathway.  Gould A, Missailidis S.  Mini Rev Med Chem. 2011 Mar;11(3):200-13.