Short and Sweet

Yamanaka strikes again. Citrulline is deiminated arginine, replacing a C=N-H (the imine) by a carbonyl C=O. An enzyme called PAD4 does the job. Why is it important? Because one of its targets is the H1 histone which links nucleosomes together. Recall that the total length of DNA in each and every one of our cells is 3 METERS. By wrapping the double helix around nucleosomes, the DNA is shortened by one order of magnitude.

So what? Well, at physiologic pH the imine probably binds another proton making it positively charged, making it bind to the negatively charged DNA phosphate backbone. Removing the imine makes this less likely to happen, so the linker doesn’t bind the double helix as tightly.

Duck soup for the chemist, but apparently no one had thought to look at this before.

This opens up the DNA (aka chromatin decondensation) for protein transcription. Why is Yamanaka involved? Because PAD4 is induced during cellular reprogramming to induced pluripotent stem cells (iPSCs), activating the expression of key stem cell genes. Inhibition of PAD4 lowers the percentage of pluripotent stem cells, reducing reprogramming efficiency. The paper is Nature vol. 507 pp. 104 – 108 ’14.

Will this may be nice for forming iPSCs, it should be noted that PAD4 is unregulated in a variety of tumors.

Curioser and curioser

Curious Wavefunction alluded to the first example of a protein which stands everything we thought we knew about them on its head. At the end of this post you’ll find another equally counterintuitive example.

We all know that proteins fold into a relatively dry core where hydrocarbon side chains and other hydrophobic elements hide out. This was one of Walter Kauzmann’s many contributions to chemistry and biology. He also wrote one of the first books on quantum chemistry, as did his PhD advisor Henry Eyring at Princeton (I was lucky enough to take PChem from him). The driving force for the formation of globular proteins according to him, was pretty much entropic, with hydrocarbon side chains solvating each other so water wouldn’t have to form an elaborate (hence structured) cage to do so.

Which brings us to the wonderfully named fish Pseudopleuronectes Americanus which lives in frigid polar waters. To keep ice crystals from forming in their cells, arctic fish have evolved proteins to prevent it. It is a fascinating example of evolution solving a problem different ways, because by 1996 at least 4 different types of antifreeze proteins were known [ PNAS vol. 93 pp. 6835 - 6840 '96 ].

The new protein is a 3 kiloDalton alanine rich helix bundle 145 Angstroms long.
Amazingly the helices surround a core of 400 water molecules (surround as in the water is on the inside of the protein, not the outside). The water molecules inside the protein are arranged as pentagons (not hexagons as they would be in ice) — so they form a clathrate. The pentagonal arrangement of water was predicted on theoretical grounds 50 years ago by Scheraga ( J. Biol. Chem. vol. ?? pp. 2506 – 2508 1962 ).

The protein has an amino acid periodicity of 11 amino acids, which nicely comes out to 3 turns of the alpha helix. There is a threonine at position i, alanine at position i + 4 and alanine a position i + 8. All of these bind water — not surprising for threonine, but alanine is a hydrocarbon. The evolving fish clearly didn’t listen to protein chemists. However, most of carbonyl groups of the protein backbone are involved in hydrogen bonding to water.

Not to be outdone, a freeze tolerant beetle (Upis cermaboides — don’t you love these names) has an antifreeze molecule made mostly of sugar and lipid.

Well even if we don’t know what we thought we knew about proteins, at least we understand biologic membranes and the proteins that go through them. Don’t we?

Apparently not. [ Proc. Natl. Acad. Sci. vol. 111 pp. 2425 - 2430 '14 ] studied the alpha-hemolysin of staphylococci. We know that the membrane of our cells is made of a double layer of molecules which a charged head which binds water and a long (16 + carbons) hydrocarbon tail. So the hydrocarbon core is 30 Angstroms across, and the lipid head groups are about 40 Angstroms away from each other on either side of the membrane.

We also know how proteins fit into the membrane — one model is the G Protein Coupled Receptor (GPCR) for which we have at least 800 human genes, and which is the target for 30% of all drugs approved by the FDA [ Science vol. 335 pp. 1106 - 1110 '12 ]. These all have 7 alpha helices arranged like a stack of logs extending across the membrane. The amino acids here are usually hydrophobic. Another model is the beta barrel — used mostly by bacteria — these have beta strands arranged across the membrane (like the staves of a barrel — get it). I’m not sure what the record is for the number of strands, but one from the gonococcus has 16 of them. They surround a large pore.

Back to the alpha hemolysin of staphylococci It’s designed to kill its target by forming a hole in the membrane. And so 7 of them get together to do so. However, instead of the running back and forth across the 30 Angstroms of the anhydrous part of the membrane, the heptamers put their heads together forming the hole (like skydivers holding hands), with their hydrocarbon like parts sticking out into the membrane and the water filled hole in the center. How do they know? They studied truncated mutants of the hemolysin, which weren’t long enough to span the 30 Angstroms across the membrane, and they still formed holes. An entirely new (to me) protein arrangement.

The Ukraine

“Are you Russian?” I asked (age 10) on meeting the formidable Dr. Antyn Rudnytsky, my future piano teacher for the first time. I then received a frightening, lengthy and intense lecture concerning the difference between Ukranians such as himself and Russians (gangsters as he called them).

What he was doing on a chicken farm in southern New Jersey in the late 40′s is quite a story. I was incredibly fortunate to have been taught by an individual of his caliber, and at amateur chamber music festivals, usually someone asks me where I’d studied. I was extremely well taught, and I spent my senior year in high school studying just the first movement of Bach’s Italian Concerto.

I have no way of checking the accuracy of all of this, but this is what I heard about him. He had a PhD in music and had studied Piano under Artur Schnabel. He was, at one point conductor of the Ukranian State Orchestra, and didn’t like the way a particular violinist played and chewed him out. The violinist denounced him to his party cell, and Dr. Rudnytsky saw his name in the paper as Mr. Rudnitsky (not Comrade Rudnytsky or even Dr. Rudnytsky). He got out and came to the USA. It took him several years to get his wife (an opera singer) and his two boys out of the Ukraine.

He never quite adjusted to the USA, speaking of how people would wait for hours in the snow to go a great concert back there and how little respect classical music had in the USA. What really must have torn him up was seeing one son (Dorian) go to Julliard, and found the New York Rock and Roll Ensemble in the 60′s where he played cello along with two guitars and a clarinet. Leonard Bernstein plugged the group for a time, ignoring the father.

His other son, Roman, was very useful to me, in that he showed me what real musical talent was like, so that I didn’t get inflated ideas about my own ability (I’m a not-too-bad amateur). At age 3 he started telling his father what notes passing trains were emitting. Then when people would come over to the house for lessons, Roman would sit behind a door, and then play what they had played (without looking at any music) on the piano. Also a Julliard graduate.

Addendum 4 Mar ’14 — I sent a copy of this post to both sons — Roman and Dorian, and almost immediately got back a nice note from Roman. Just Google him (Roman Rudnytsky) for some of his U-Tubes etc. He said that everything I remembered about his father and his history was ‘spot on’.

One more Ukrainian bit before moving on to the present. In the 80s a newly arrived Ukranian lady was interviewed by the local paper in upstate NY. When asked what she liked about the US, she mentioned having people over to her house for prayer without having to draw the shades.

So now Russia has invaded the Crimea again, and Europe is reduced to making a few noises. Since they spend about 20% as much as the USA on defense, it’s about all they can do (but look at the great social services they have — they won’t be much help if Russia moves west again).

Another even more disturbing point, is that we talked Ukraine into giving up its nuclear weapons. In June 1996 they transferred all 1,900 of their nuclear weapons to Russia. It is very doubtful that Russia would have invaded, had the Ukraine retained them. It is even more doubtful, that any country with nuclear weapons will ever again voluntarily give them up. It is also quite likely that many small countries without them will try to go nuclear. The world has just become a much more dangerous place.

On the bright side, Europeans can now put their large numbers of unemployed youth into their armies, solving at least one problem.

Two very scary papers about cancer

What if, even after you’ve killed every cancer cell in the body, there are still non-malignant cells left that are halfway there. That’s the conclusion of two very scary papers published in the past week[ Nature vol. 506 pp, 300 - 301, 328 - 33 '14, Proc. Natl. Acad. Sci. vol. 111 pp. 2548 - 2553 '14 ]. Both involve acute myelogenous leukemia (AML).

Blood cancers are easy to study even without getting samples of the marrow, which is (relatively) easy to come by. The marrow contains stem cells which can form all the cellular elements of blood (red cells, all types of white cells and platelets). They are called hematopoietic stem cells (HSCs), and just one of them is enough to completely repopulate the radiation destroyed marrow of an experimental animal.

Even a person suffering with AML contains functionally normal HSCs in their marrow (otherwise they’d be dead). What these papers show, is that these cells contain some, but not all of the mutations found in the leukemic cells (their names are DNMT3A, IDH1, IDH2, ASXL1, IKZK1 — they don’t roll trippingly off the tongue do they?). They are called preleukemic cells, and the papers show that conventional therapy for AML does NOT kill them. Essentially these cells are accidents waiting to happen.

The PNAS paper calls these genes ‘landscaping genes’, a term which may be original. I love the term, it’s extremely descriptive and short. These are genes involved in global chromatin changes — we’re talking epigenetics here — proteins causing changes in DNA and the proteins that bind to it, which don’t actually change the order of bases in the DNA.

Hopefully this doesn’t apply to other forms of cancer, but I have a sinking feeling that it does. So getting rid of every cancer cell in the body, may not be enough. Frightening.

Are memories stored outside of neurons?

This may turn out to be a banner year for neuroscience. Work discussed in the following older post is the first convincing explanation of why we need sleep that I’ve seen.

An article in Science (vol. 343 pp. 670 – 675 ’14) on some fairly obscure neurophysiology at the end throws out (almost as an afterthought) an interesting idea of just how chemically and where memories are stored in the brain. I find the idea plausible and extremely surprising.

You won’t find the background material to understand everything that follows in this blog. Hopefully you already know some of it. The subject is simply too vast, but plug away. Here a few, seriously flawed in my opinion, theories of how and where memory is stored in the brain of the past half century.

#1 Reverberating circuits. The early computers had memories made of something called delay lines ( where the same impulse would constantly ricochet around a circuit. The idea was used to explain memory as neuron #1 exciting neuron #2 which excited neuron . … which excited neuron #n which excited #1 again. Plausible in that the nerve impulse is basically electrical. Very implausible, because you can practically shut the whole brain down using general anesthesia without erasing memory.

#2 CaMKII — more plausible. There’s lots of it in brain (2% of all proteins in an area of the brain called the hippocampus — an area known to be important in memory). It’s an enzyme which can add phosphate groups to other proteins. To first start doing so calcium levels inside the neuron must rise. The enzyme is complicated, being comprised of 12 identical subunits. Interestingly, CaMKII can add phosphates to itself (phosphorylate itself) — 2 or 3 for each of the 12 subunits. Once a few phosphates have been added, the enzyme no longer needs calcium to phosphorylate itself, so it becomes essentially a molecular switch existing in two states. One problem is that there are other enzymes which remove the phosphate, and reset the switch (actually there must be). Also proteins are inevitably broken down and new ones made, so it’s hard to see the switch persisting for a lifetime (or even a day).

#3 Synaptic membrane proteins. This is where electrical nerve impulses begin. Synapses contain lots of different proteins in their membranes. They can be chemically modified to make the neuron more or less likely to fire to a given stimulus. Recent work has shown that their number and composition can be changed by experience. The problem is that after a while the synaptic membrane has begun to resemble Grand Central Station — lots of proteins coming and going, but always a number present. It’s hard (for me) to see how memory can be maintained for long periods with such flux continually occurring.

This brings us to the Science paper. We know that about 80% of the neurons in the brain are excitatory — in that when excitatory neuron #1 talks to neuron #2, neuron #2 is more likely to fire an impulse. 20% of the rest are inhibitory. Obviously both are important. While there are lots of other neurotransmitters and neuromodulators in the brains (with probably even more we don’t know about — who would have put carbon monoxide on the list 20 years ago), the major inhibitory neurotransmitter of our brains is something called GABA. At least in adult brains this is true, but in the developing brain it’s excitatory.

So the authors of the paper worked on why this should be. GABA opens channels in the brain to the chloride ion. When it flows into a neuron, the neuron is less likely to fire (in the adult). This work shows that this effect depends on the negative ions (proteins mostly) inside the cell and outside the cell (the extracellular matrix). It’s the balance of the two sets of ions on either side of the largely impermeable neuronal membrane that determines whether GABA is excitatory or inhibitory (chloride flows in either event), and just how excitatory or inhibitory it is. The response is graded.

For the chemists: the negative ions outside the neurons are sulfated proteoglycans. These are much more stable than the proteins inside the neuron or on its membranes. Even better, it has been shown that the concentration of chloride varies locally throughout the neuron. The big negative ions (e.g. proteins) inside the neuron move about but slowly, and their concentration varies from point to point.

Here’s what the authors say (in passing) “the variance in extracellular sulfated proteoglycans composes a potential locus of analog information storage” — translation — that’s where memories might be hiding. Fascinating stuff. A lot of work needs to be done on how fast the extracellular matrix in the brain turns over, and what are the local variations in the concentration of its components, and whether sulfate is added or removed from them and if so by what and how quickly.

We’ve concentrated so much on neurons, that we may have missed something big. In a similar vein, the function of sleep may be to wash neurons free of stuff built up during the day outside of them.

The weirdness of gravity

We experience gravity every waking moment, so it’s hard to recognize just how strange the gravitational ‘force’ actually is. Push a toy sailboat, a rowboat, and a yacht with the same amount of force (effort). What happens?

The smaller the boat, the faster it moves. Physicists would say the acceleration (change in velocity over time e.g. from the boat not moving at all to moving somewhat) is inversely proportional to the mass of the boat. This is Newton’s famous second law force = mass * acceleration. This isn’t actually what he said which you’ll find at the end.

So in every force except gravity, the bigger the force the more the acceleration. In Galileo’s famous experiment (which Wikipedia says might actually not have occurred), he dropped 2 objects of different masses from the leaning tower of Pisa and found that they hit the ground at the same time, so the acceleration of both due to the ‘force’ of gravity is the for all objects regardless of their different masses.

This implies that gravity is a force that adjusts itself to the mass of the object it is pushing on to produce the same acceleration. Weird, but true.

General relativity says, that the motion must be considered not just in space and time, but in 4 dimensional space-time where space can become our conventional time and vice versa. Here all paths are as straight as possible — because the 4 dimensional space-time we inhabit has an intrinsic curvature, produced by the masses found within it.

What Newton said: “The change of motion is proportional to the motive force impressed and is made in the direction of the straight line in which that force is impressed” By motion Newton means what we call momentum — mass * velocity.

The change in momentum is of course a change in velocity — which is what acceleration actually is. Note that mass is assumed constant regardless of how fast the object is moving. This isn’t even true in special relativity (which doesn’t include gravity — that’s what general relativity is all about).

The Silence of the Times

This just in — Ramallah occupied territories — Israeli Defense Forces killed 3 students protesting the occupation.

Don’t you think this would be on the front page of the New York Times, The Washington Post, all over CNN and MSNBC.

On 13 Feb the Times noted that 2 students protesting in Venezuela had been killed (3 actually). Absolutely nothing further about it, or the protests. Nothing to see here. Move on sayeth the NYT and the Boston Globe as far as I can tell.

They like to think of themselves as the fearless, investigative press.

Everything not expressly forbidden biochemically is happening somewhere

A fairly oblique introduction (from an earlier post)

Sherlock Holmes and the Green Fluorescent Protein

Gregory (Scotland Yard): “Is there any other point to which you would wish to draw my attention?”
Holmes: “To the curious incident of the dog in the night-time.”
Gregory: “The dog did nothing in the night-time.”
Holmes: “That was the curious incident.”

The chromophore of green fluorescent protein (GFP) is para-hydroxybenzylidene imidazolinone. It is formed by cyclization of a serine (#65) tyrosine (#66) glycine (#67) sequential tripeptide. It is found in the center of a beta barrel formed by the 238 amino acids of GFP.

What is so curious about this?

Simply put, why don’t things like this happen all the time? Perhaps nothing quite this fancy, but on a more plebeian level consider this: of the twenty amino acids, 2 are carboxylic acids, 2 are amides, 1 is an amine, 3 are alcohols and one is a thiol. One might expect esters, amides, thioesters and sulfides to be formed deep inside proteins. Why deep inside? On the surface of the protein, there is water at 55 molar around to hydrolyze them purely by the law of mass action (releasing about 10 kJ/Avogadro’s number per bond in the process). Some water is present in the X-ray crystallographic structure of proteins, but nothing this concentrated.

The presence of 55 M water bathing the protein surface leads to an even more curious incident, namely why proteins exist at all given that amide hydrolysis is exothermic (as well as entropically favorable). Perhaps this is why proteins contain so many alpha helices and beta sheets — as well as functioning as structural elements they may also serve to hide the amides from water by hydrogen bonding them to each other. Along this line, could this be why the hydrophilic side chains of proteins (arginine, lysine, the acids and the amides) are rather bulky? Perhaps they also function to sterically shield the adjacent amides. After all, why should lysine have 4 CH2 groups to separate the primary amino from the alpha carbon? Ditto for the 3 CH2 groups separating the guanidine group, and the 2 CH2 for glutamic acid.

We now have an example before us of an ester between threonine and glutamic acid within the same protein. For details see Proc. Natl. Acad. Sci. vol. 111 pp. 1229 – 1230, 1367 – 1372 ’14. It is put to use to stabilize long thin proteins subject to mechanical stress. All sorts have bacteria have little hairs (pili) allowing them to attach to our cells. The first example were found in some nasty characters (Streptococcus progenies, Clostridium perfringens), possibly because they’re under intense study because the infections they cause are even nastier. Interestingly, the ester is buried deep in the protein where water can’t get at it so easily. This type of link on external proteins turns out to be fairly common in Gram positive organisms.

So everything not biochemically forbidden is probably happening somewhere.

Here’s just how poor the research on Autism Spectrum Disorder has been

A stroke 50 years ago would be a stroke today. Cancer is still cancer. Not so with Autism, Autism Spectrum Disorder (ASD), Asperger’s syndrome. The Diagnostic and Statistical Manual of the American Psychiatric Association (DSM to you) has gone through 6 editions (DSM-I through DSM-V, including DSM-IV-TR). The terms are defined differently in all (or not even defined). For the gory details see:

Here’s a summary.

DSM-I (1952) & DSM-II (1968)
No term Autism or Pervasive Developmental Disorder Closest term: Schizophrenic Reaction (Childhood Type)
1980 DSM-III
Pervasive Developmental Disorders (PDD):
Childhood Onset PDD, Infantile Autism, Atypical Autism
1987 DSM-III-R
Pervasive Developmental Disorders (PDD):
PDD-NOS, Autistic Disorder
1994 DSM-IV
Pervasive Developmental Disorders (PDD):
PDD-NOS, Autistic Disorder, Asperger Disorder, Childhood Disintegrative Disorder, Rett syndrome
2000 DSM-IV-TR
Same diagnoses, text correction for PDD-NOS

Needless to say, DSM-V (out since May 2013) has it differently.

The following paper [ Proc. Natl. Acad. Sci. vol. 111 pp. 1981 - 1986 '14 ] should have neuroscientific researchers on Autism Spectrum Disorder (ASD) of the DSM-IV hanging their heads in shame.

The criteria for ASD are obviously a wastebasket. Here they are from the DSM-IV-TR

Autistic disorder (classic autism)
Asperger’s disorder (Asperger syndrome)
Pervasive developmental disorder not otherwise specified (PDD-NOS)
Rett’s disorder (Rett syndrome)
Childhood disintegrative disorder (CDD).

Just about any kid not doing well cognitively fits in to #3. Hopefully the DSM-V makes things better — it’s too early to tell. The link has the details and a lot of the reasoning behind yet another change.

All the work cited in the PNAS paper concerns ASD as diagnosed by DSM-IV-TR. DSM-V is too new to have papers out using its criteria.

If you take 100 kids developing normally, do various types of magnetic resonance imaging (MRI) on their brains, and compare then with 100 kids not doing well, you are certain to find more structural abnormalities of the brain in the second group, regardless of how they were diagnosed, or what they were diagnosed with.

Dozens of papers were written on MRIs in ASD kids. 40% had fewer than 15 subjects. The most replicated finding (up to the PNAS paper) was poor connections between various parts of the brain, manifest as abnormalities of the white matter. Exactly what they were measuring, and how the measurement requires a tensor and not a vector is really quite interesting and can teach you some math. I’ll save this for the end.

Only 2 of the dozens of papers controlled for data quality. MRIs have been around long enough, that most know that to get a decent study the subject has to be quite still, something very difficult for kids, and even harder for autistic kids.

When head motion was controlled for in this large (52 ASDs 73 normals to start) all the abnormalities disappeared (save one, and they looked at 18 different white matter tracts in the brain). They had to throw out the studies on 12/52 of the autistics and 2/73 normals because of motion– showing how suspect the previous data really was. The head motion was producing the abnormalities.

Is this terrible research or what (PNAS paper excepted)?

Perhaps the new criteria in DSM-V will result in a more homogenous group.

What does diffusion tensor imaging actually measure? Imagine the nerve fibers (axons) of the brain in the deep white matter as a bundle of wires, most of them going in the same direction in any small volume. Assume they are bathed in water. Now add some colored water at one end, and see how quickly the color diffuses in various directions in the bundle. The color will diffuse faster along the bundle, than in the two directions perpendicular to it. This is what the diffusion tensor measures — diffusion of tissue water in a variety of directions. If the bundle is loose, or disorganized, or some wires are missing, than the diffusion in the various directions won’t differ as much — this is called lack of anisotropy. Take out the wires and the diffusion is the same in all directions (no anisotropy at all). This was the finding in ASD — less white matter anisotropy in diffusion tensor imaging — implying that there is something wrong with it.

Why wouldn’t an overall vector summing up the diffusion in the major direction be enough. One can add vectors together after all. Because you’d lose all the information about anisotropy. The tensor preserves it. It’s why tensors are used to measure the stress in a given material. Slick. Now you understand (something) about tensors. However it should be noted that vectors are tensors too. There’s a lot more too it (particularly indices).

While reading the research literature is a joy, sometimes it isn’t

“In this sense, enhanced connectivity of an essential node [e.g., in this study, as suggested by the previous analysis by Wühle et al. (17), the secondary somatosensory cortex, S2; for the issue of whether S1 is an essential node, see ref. 22] to brain structures, which render information consciously accessible, constitute predefined or privileged pathways along which neural information can propagate when confronted with an appropriate stimulus.”

I won’t tell you where this is from, but it’s but one horrible sentence among many. Even worse, the paper, is about something quite interesting — how much of the brain (and which parts) have to be activated before a barely perceptible stimulus is reported (e.g. when and where does consciousness begin).


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