Category Archives: Medicine in general

The perfect aphrodisiac ?

We’re off to London for a few weeks to celebrate our 50th Wedding Anniversary. As a parting gift to all you lovelorn organic chemists out there, here’s a drug target for a new aphrodisiac.

Yes, it’s yet another G Protein Coupled Receptor (GPCR) of which we have 800+ in our genome, and which some 30% of drugs usable in man target (but not this one).

You can read all about it in a leisurely review of “Affective Touch” in Neuron vol. 82 pp. 737 – 755 ’14, and Nature vol. 493 pp. 669 – 673 ’13. The receptor (if the physiological ligand is known the papers are silent about it) is found on a type of nerve going to hairy skin. It’s called MRGPRB4.

The following has been done in people. Needles were put in a cutaneous nerve, and skin was lightly stroked at rates between 1 and 10 centimeters/second. Some of the nerves respond at very high frequency 50 – 100 impulses/second (50 – 100 Hertz) to this stimulus. Individuals were asked to rate the pleasantness of the sensation produced. The most pleasant sensations produced the highest frequency responses of these nerves.

MRGPRB4 is found on nerves which respond like this (and almost nowhere else as far as is known), so a ligand for it should produce feelings of pleasure. The whole subject of proteins which produce effects when the cell carrying them is mechanically stimulated is fascinating. Much of the work has been done with the hair cells of the ear, which discharge when the hairs are displaced by sound waves. Proteins embedded in the hairs trigger an action potential when disturbed.

Perhaps there is no chemical stimulus for MRGPRB4, just as there isn’t for the hair cells, but even so it’s worth looking for some chemical which does turn on MRGPRB4. Perhaps a natural product already does this, and is in one of the many oils and lotions people apply to themselves. Think of the chemoattractants for bees and other insects.

If you’re the lucky soul who finds such a drug, fame and fortune (and perhaps more) is sure to be yours.

Happy hunting

Back in a few weeks

A huge amount of work will need to be redone

The previous post is reprinted below the —- if you haven’t read it, you should do so now before proceeding.

Briefly, no one had ever bothered to check if subjects were asleep while studying the default mode of brain activity. The paper discussed in the previous post appeared in the 7 May ’14 issue of Neuron.

In the 13 May ’14 issue of PNAS [ Proc. Natl. Acad. Sci. vol. 111 pp. E2066 - E2075 '14 ] a paper appeared on genetic links to default mode abnormalities in schizophrenia and bipolar disorder.

From the abstract “Study subjects (n = 1,305) underwent a resting-state functional MRI scan and were analyzed by a two-stage approach. The initial analysis used independent component analysis (ICA) in 324 healthy controls, 296 Schizophrenic probands, 300 psychotic bipolar disorder probands, 179 unaffected first-degree relatives of schizophrenic pro bans, and 206 unaffected first-degree relatives of psychotic bipolar disorder probands to identify default mode networks and to test their biomarker and/or endophenotype status. A subset of controls and probands (n = 549) then was subjected to a parallel ICA (para-ICA) to identify imaging–genetic relationships. ICA identified three default mode networks.” The paper represents a tremendous amount of work (and expense).

No psychiatric disorder known to man has normal sleep. The abnormalities found in the PNAS study may not be of the default mode network, but in the way these people are sleeping. So this huge amount of work needs to be repeated. An tghis is just one paper. As mentioned a Google search on Default Networks garnered 32,000,000 hits.

Very sad.

____

How badly are thy researchers, O default mode network

If you Google “default mode network” you get 32 million hits in under a second. This is what the brain is doing when we’re sitting quietly not carrying out some task. If you don’t know how we measure it using functional mMRI skip to the **** and then come back. I’m not a fan of functional MRI (fMRI), the pictures it produces are beautiful and seductive, and unfortunately not terribly repeatable.

If [ Neuron vol. 82 pp. 695 - 705 '14 ] is true than all the work on the default network should be repeated.

Why?

Because they found that less than half of 71 subjects studied were stably awake after 5 minutes in the scanner. E.g. they were actually asleep part of the time.

How can they say this?

They used Polysomnography — which simultaneously measures tons of things — eye movements, oxygen saturation, EEG, muscle tone, respiration pulse; the gold standard for sleep studies on the patients while in the MRI scanner.

You don’t have to be a neuroscientist to know that cognition is rather different in wake and sleep.

Pathetic.

****

There are now noninvasive methods to study brain activity in man. The most prominent one is called BOLD, and is based on the fact that blood flow increases way past what is needed with increased brain activity. This was actually noted by Wilder Penfield operating on the brain for epilepsy in the 30s. When the patient had a seizure on the operating table (they could keep things under control by partially paralyzing the patient with curare) the veins in the area producing the seizure turned red. Recall that oxygenated blood is red while the deoxygenated blood in veins is darker and somewhat blue. This implied that more blood was getting to the convulsing area than it could use.

BOLD depends on slight differences in the way oxygenated hemoglobin and deoxygenated hemoglobin interact with the magnetic field used in magnetic resonance imaging (MRI). The technique has had a rather checkered history, because very small differences must be measured, and there is lots of manipulation of the raw data (never seen in papers) to be done. 10 years ago functional magnetic imaging (fMRI) was called pseudocolor phrenology.

Some sort of task or sensory stimulus is given and the parts of the brain showing increased hemoglobin + oxygen are mapped out. As a neurologist, I was naturally interested in this work. Very quickly, I smelled a rat. The authors of all the papers always seemed to confirm their initial hunch about which areas of the brain were involved in whatever they were studying. Science just isn’t like that. Look at any issue of Nature or Science and see how many results were unexpected. Results were largely unreproducible. It got so bad that an article in Science 2 August ’02 p. 749 stated that neuroimaging (e.g. functional MRI) has a reputation for producing “pretty pictures” but not replicable data. It has been characterized as pseudocolor phrenology (or words to that effect).

What was going on? The data was never actually shown, just the authors’ manipulation of it. Acquiring the data is quite tricky — the slightest head movement alters the MRI pattern. Also the difference in NMR signal between hemoglobin without oxygen and hemoglobin with oxygen is small (only 1 – 2%). Since the technique involves subtracting two data sets for the same brain region, this doubles the error.

How badly are thy researchers, O default mode network

If you Google “default mode network” you get 32 million hits in under a second. This is what the brain is doing when we’re sitting quietly not carrying out some task. If you don’t know how we measure it using functional mMRI skip to the **** and then come back. I’m not a fan of functional MRI (fMRI), the pictures it produces are beautiful and seductive, and unfortunately not terribly repeatable.

If [ Neuron vol. 82 pp. 695 - 705 '14 ] is true than all the work on the default network should be repeated.

Why?

Because they found that less than half of 71 subjects studied were stably awake after 5 minutes in the scanner. E.g. they were actually asleep part of the time.

How can they say this?

They used Polysomnography — which simultaneously measures tons of things — eye movements, oxygen saturation, EEG, muscle tone, respiration pulse; the gold standard for sleep studies on the patients while in the MRI scanner.

You don’t have to be a neuroscientist to know that cognition is rather different in wake and sleep.

Pathetic.

****

There are now noninvasive methods to study brain activity in man. The most prominent one is called BOLD, and is based on the fact that blood flow increases way past what is needed with increased brain activity. This was actually noted by Wilder Penfield operating on the brain for epilepsy in the 30s. When the patient had a seizure on the operating table (they could keep things under control by partially paralyzing the patient with curare) the veins in the area producing the seizure turned red. Recall that oxygenated blood is red while the deoxygenated blood in veins is darker and somewhat blue. This implied that more blood was getting to the convulsing area than it could use.

BOLD depends on slight differences in the way oxygenated hemoglobin and deoxygenated hemoglobin interact with the magnetic field used in magnetic resonance imaging (MRI). The technique has had a rather checkered history, because very small differences must be measured, and there is lots of manipulation of the raw data (never seen in papers) to be done. 10 years ago functional magnetic imaging (fMRI) was called pseudocolor phrenology.

Some sort of task or sensory stimulus is given and the parts of the brain showing increased hemoglobin + oxygen are mapped out. As a neurologist, I was naturally interested in this work. Very quickly, I smelled a rat. The authors of all the papers always seemed to confirm their initial hunch about which areas of the brain were involved in whatever they were studying. Science just isn’t like that. Look at any issue of Nature or Science and see how many results were unexpected. Results were largely unreproducible. It got so bad that an article in Science 2 August ’02 p. 749 stated that neuroimaging (e.g. functional MRI) has a reputation for producing “pretty pictures” but not replicable data. It has been characterized as pseudocolor phrenology (or words to that effect).

What was going on? The data was never actually shown, just the authors’ manipulation of it. Acquiring the data is quite tricky — the slightest head movement alters the MRI pattern. Also the difference in NMR signal between hemoglobin without oxygen and hemoglobin with oxygen is small (only 1 – 2%). Since the technique involves subtracting two data sets for the same brain region, this doubles the error.

Why marihuana scares me

There’s an editorial in the current Science concerning how very little we know about the effects of marihuana on the developing adolescent brain [ Science vol. 344 p. 557 '14 ]. We know all sorts of wonderful neuropharmacology and neurophysiology about delta-9 tetrahydrocannabinol (d9-THC) — http://en.wikipedia.org/wiki/Tetrahydrocannabinol The point of the authors (the current head of the Amnerican Psychiatric Association, and the first director of the National (US) Institute of Drug Abuse), is that there are no significant studies of what happens to adolescent humans (as opposed to rodents) taking the stuff.

Marihuana would the first mind-alteraing substance NOT to have serious side effects in a subpopulation of people using the drug — or just about any drug in medical use for that matter.

Any organic chemist looking at the structure of d9-THC (see the link) has to be impressed with what a lipid it is — 21 carbons, only 1 hydroxyl group, and an ether moiety. Everything else is hydrogen. Like most neuroactive drugs produced by plants, it is quite potent. A joint has only 9 milliGrams, and smoking undoubtedly destroys some of it. Consider alcohol, another lipid soluble drug. A 12 ounce beer with 3.2% alcohol content has 12 * 28.3 *.032 10.8 grams of alcohol — molecular mass 62 grams — so the dose is 11/62 moles. To get drunk you need more than one beer. Compare that to a dose of .009/300 moles of d9-THC.

As we’ve found out — d9-THC is so potent because it binds to receptors for it. Unlike ethanol which can be a product of intermediary metabolism, there aren’t enzymes specifically devoted to breaking down d9-THC. In contrast, fatty acid amide hydrolase (FAAH) is devoted to breaking down anandamide, one of the endogenous compounds d9-THC is mimicking.

What really concerns me about this class of drugs, is how long they must hang around. Teaching neuropharmacology in the 70s and 80s was great fun. Every year a new receptor for neurotransmitters seemed to be found. In some cases mind benders bound to them (e.g. LSD and a serotonin receptor). In other cases the endogenous transmitters being mimicked by a plant substance were found (the endogenous opiates and their receptors). Years passed, but the receptor for d9-thc wasn’t found. The reason it wasn’t is exactly why I’m scared of the drug.

How were the various receptors for mind benders found? You throw a radioactively labelled drug (say morphine) at a brain homogenate, and purify what it is binding to. That’s how the opiate receptors etc. etc. were found. Why did it take so long to find the cannabinoid receptors? Because they bind strongly to all the fats in the brain being so incredibly lipid soluble. So the vast majority of stuff bound wasn’t protein at all, but fat. The brain has the highest percentage of fat of any organ in the body — 60%, unless you considered dispersed fatty tissue an organ (which it actually is from an endocrine point of view).

This has to mean that the stuff hangs around for a long time, without any specific enzymes to clear it.

It’s obvious to all that cognitive capacity changes from childhood to adult life. All sorts of studies with large numbers of people have done serial MRIs children and adolescents as the develop and age. Here are a few references to get you started [ Neuron vol. 72 pp. 873 - 884, 11, Proc. Natl. Acad. Sci. vol. 107 pp. 16988 - 16993 '10, vol. 111 pp. 6774 -= 6779 '14 ]. If you don’t know the answer, think about the change thickness of the cerebral cortex from age 9 to 20. Surprisingly, it get thinner, not thicker. The effect happens later in the association areas thought to be important in higher cognitive function, than the primary motor or sensory areas. Paradoxical isn’t it? Based on animal work this is thought to be due pruning of synapses.

So throw a long-lasting retrograde neurotransmitter mimic like d9-THC at the dynamically changing adolescent brain and hope for the best. That’s what the cited editorialists are concerned about. We simply don’t know and we should.

Having been in Cambridge when Leary was just getting started in the early 60’s, I must say that the idea of tune in turn on and drop out never appealed to me. Most of the heavy marihuana users I’ve known (and treated for other things) were happy, but rather vague and frankly rather dull.

Unfortunately as a neurologist, I had to evaluate physician colleagues who got in trouble with drugs (mostly with alcohol). One very intelligent polydrug user MD, put it to me this way — “The problem is that you like reality, and I don’t”.

Is a sea change taking place at the New York Times ?

The little kid started crying as I approached him with the syringe filled with yellow fluid. He knew that after he was held down and I injected him he would be violently sick and vomit repeatedly.

It was 1964 and this happened at the Children’s Hospital of Philadelphia (CHOP) and the kid had acute lymphatic leukemia, and the syringe was full of methotrexate, the antifolate drug in use at the time. I was a third year med student. Although Stanley Milgram had begun his “Obedience to Authority” experiments in 1961 http://en.wikipedia.org/wiki/Milgram_experiment, I was hardly a happy or willing participant in the proceedings. I had nightmares about it.

Like all the kids with leukemia at CHOP, the little boy was part of a ‘study’ run by an oncologist, with an accent right out of Boris Karloff. I thought he was a monster. He was so happy that the kids in his branch of the study survived a horrible 21 months, vs. the previous record of 18. I thought that the kids were being kept alive and suffering when they shouldn’t have been, in order to set a new survival record. The study randomized the kids between the new regimen and the current regimen showing the best survival.

Well, I was terribly wrong, and the oncologist was a hero not a monster. Presently the cure rate (not survival) of childhood leukemia is over 90%. We now worry about the long term side effects of the drugs (and radiation) used to cure it — cognitive problems, fertility problems. It was precisely because the new treatment was compared to the best previous treatment that we are where we are today.

What in the world does this have to do with the New York Times?

Simply this, on Monday 21 April the front page of the New York Times contained an article title “50 Years Later, Hardship Hits Back, Poorest Counties Are Still Losing in War on Want”. They don’t call it the “War on Poverty” until the 5th paragraph. Nonetheless, the article (without explicitly saying so) documents just what a failure it has been. Nowhere in the article, is there any mention of why it failed, but it’s clear that only more of the same has been tried — more food stamps, more medicaid, more free school lunches, etc. etc. It is claimed in the article that this lifted tens of thousands above a subsistence standard of living, yet 15% of the populace is still living in poverty and 47/300 million of us are on food stamps.

At least the Times is no longer pretending that the War on Poverty (started in 1964 when I was pushing methotrexate) is a success.

Another sign of a sea change at the Times appeared the day before on the Op-Ed page in an article titled “From Rags to Riches to Rags” in which the notion of a static top 1% in income was debunked. A study of 44 years of longitudinal data of people from 25 to 60 showed that 12% of all of them would be in the top 1% of income for at least one year, and that 39% will be in the top 5% of income for at least 1 year.

A third appeared on the 22nd in a front page article concerning a near lynching by Blacks in Detroit of a white man who hit a child with his car.

In recent years, I’ve thought that I’ve had to read the Times much as the Russians read Pravda during cold war I (and perhaps today). A friend has called it ‘advocacy driven journalism’. Perhaps there will be a shift in orientation from left to right, but, even so, I’m not a fan of having articles #1 and #3 any place other than Op – Ed page. Advocacy journalism is advocacy journalism whether it agrees with your political orientation or not. The 3 articles cited really aren’t news. That’s what the opinion page is for — opinion and background.

80+ years ago my future parents discovered that one of the first things they had in common was that they both read the Times. I grew up with it, and hopefully it will become a great newspaper again.

The failure to try anything new against poverty is a manifestation of the arrogance of the intelligent, about which there will be another post.

Further (physical) chemical elegance

If the chemical name phosphatidyl serine (PS) draws a blank, read the verbatim copy of a previous post under the *** to find out why it is so important to our existence. It is an ‘eat me’ signal when there is lots of it around, telling professional scavenger cells to engulf the cell showing lots of PS on its surface.

Life, as usual, is more complicated. There are a variety of proteins exposed on cell surfaces which bind to phosphoserine. Not only that, but exposing just a little PS on the surface of a cell can trigger a protective immune response. Immune cells binding to just a little PS on the surface of another cell proliferate rather than eat the cell expressing the PS. This brings us to Proc. Natl. Acad. Sci. vol. 111 pp 5526 – 5531 ’14 that explains how a given PS receptor (called TIM4) acts differently depending how much PS is present.

Some PS receptors such as Annexin V have essentially an all or none response to PS, if they bind at all, they trigger a response in the cell carrying them. Not so for TIM4 which only reacts if there is a lot of PS around, leaving cells which express less PS alone. This allows these cells to function in the protective immune response.

So how does TIM4 do this? See if you can think of a mechanism before reading the rest.

In addition to the PS binding pocket TIM4 has 4 peripheral basic residues in separate places. The basic residues are positively charged at physiologic pH and bind to the negatively charged phosphate group of phosphatidyl serene or to the carboxylate anion of phosphatidyl serine. The paper doesn’t explain how these basic residues don’t bind to the other phospholipids of the cell surface (such as phosphatidyl choline or sphingomyelin). It is conceivable that the basic side chains (arginine, lysine etc.) are so set up that they only bind to carboxylate anions and not phosphate anions (but this is a stretch). That would at least give them specificity for phosphatidyl serene as opposed the other phospholipids present in both leaflets of the cell membrane. In any even TIM4 will be triggered only if these groups also bind PS, leaving cells which show relatively little PS alone. Clever no?

For the cognoscenti, the Hill coefficient of TIM4 is 2 while that of Annexin V is 8 (describing more than explaining the all or none character of Annexin V binding).

****
Flippase. Eat me signals. Dragging their tails behind them. Have cellular biologists and structural biochemists gone over to the dark side? It’s all quite innocuous as the old nursery rhyme will show

Little Bo Peep has lost her sheep
and doesn’t know where to find them
Leave them alone, and they’ll come home
wagging their tails behind them.

First, some cellular biochemistry. The lipid bilayer encasing all our cells is made of two leaflets, inner and outer. The composition of the two is different (unlike the soap bubble). On the inside we find phosphatidylethanolamine (PE), phosphatidylserine (PS). The outer leaflet contains phosphatidylcholine (PC) and sphingomyelin (SM) and almost no PE or PS. This is clearly a low entropy situation compared to having all 4 randomly dispersed between the 2 leaflets.

What is the possible use of this (notice how teleology invariably creeps into cellular biology)? Chemistry is powerless to explain such things. Much as I love chemistry, such truths must be faced.

It takes energy to maintain this peculiar distribution. The enzyme moving PE and PS back inside the cell is the flippase. It requires energy in the form of ATP to operate. When a cell is dying ATP drops, and entropy takes its course moving PE and PS to the cell surface. Specialized cells (macrophages) exist to scoop up the dying or dead cells, without causing inflammation. They recognize PE and PS by a variety of receptors and munch up cells exposing them on the surface. So PE and PS are eat me signals which appear when there isn’t enough ATP around for flippase to use to haul PE and PS back inside. Clever no?

No for some juicy chemistry (assuming that you consider transport of a molecule across a lipid bilayer actual chemistry — no covalent bonds to the transferred molecule are formed or removed, although they are to the transporter). Well it certainly is physical chemistry isn’t it?

Here are the structures of PE, PS, PC, SM http://www.google.com/search?q=phosphatidylserine&client=safari&rls=en&tbm=isch&tbo=u&source=univ&sa=X&ei=bDRLU5yfHOPLsQSOnoG4BA&ved=0CPABEIke&biw=1540&bih=887#facrc=_&imgdii=_&imgrc=qrLByG2vmhWdwM%253A%3BwAtgsTPwCxeZXM%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fimg%252Fpng%252FCommon_Phospholipids.png%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fcoursework_pages%252F2012_02_2.html%3B1297%3B934.

There are a few things to notice. Like just about every lipid found in our membranes, they are amphipathic — they have a very lipid soluble part (look at the long hydrocarbon changes hanging below them) and a very water soluble part — the head groups containing the phosphate.

This brings us to [ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 - E1343 '14 ] Which describes ATP8A2 (aka the flippase). Interestingly, the protein, with at least 10 alpha helices spanning the membrane, and 3 cytoplasmic domains closely resembles the classic sodium pump beloved of neurophysioloogists everywhere, which pumps sodium ions out of neurons and pumps potassium ions inside, producing the equally beloved membrane potential of neurons.

Look at those structures again. While there are charges on PE, PS (on the phosphate group), these molecules are far larger than the sodium or the potassium ion (easily by a factor of 10). This has long been recognized and is called the ‘giant substrate problem’.

The paper solved the structure of ATP8A2 and used molecular dynamics stimulations to try to understand how it works. What they found is that transmembrane alpha helices 1, 2, 4 and 6 (out of 10) form a water filled cavity, which dissolves the negatively charged phosphate of the head group. What happens to those long hydrocarbon tails? The are left outside the helices in the lipid core of the membrane. It is the charged head groups that are dragged through by the flippase, with the tails wagging along behind them, just like little Bo Peep.

There’s a lot more great chemistry in the paper, particularly how Isoleucine #364 directs the sequential formation and annihilation of the water filled cavities between alpha helices 1, 2, 4 and 6, and how a particular aspartic acid is phosphorylated (by ATP, explaining why the enzyme no longer works in energetically dying cells) changing conformation of all 10 transmembrane helices, so that only one half of the channel is open at a time (either to the inside or the outside).

Go read and enjoy. It’s sad that people who don’t know organic chemistry are cut off from appreciating such elegance. There is more to esthetics than esthetics.

Just when you thought you understood neurotransmission

Back in the day, the discovery of neurotransmission allowed us to think we understood how the brain worked. I remember explaining to medical students in the early 70s, that the one way flow of information from the presynaptic neuron to the post-synaptic one was just like the flow of current in a vacuum tube — yes a vacuum tube, assuming anyone reading knows what one is. Later I changed this to transistor when integrated circuits became available.

Also the Dale hypothesis as it was taught to me, was that a given neuron released the same neurotransmitter at all its endings. As it was taught back in the 60s this meant that just one transmitter was released by a given neuron.

Retrograde transmission was just a glimmer in the mind’s eye back then. We now know that the post-synaptic neuron releases compounds which affect the presynaptic neuron, the supposed controller of the postsynaptic neuron. Among them are carbon monoxide, and the endocannabinoids (e. g. what marihuana is trying to mimic).

In addition there are neurotransmitter receptors on the presynaptic neuron, which respond to what it and other neurons are releasing to control its activity. These are outside the synapse itself. These events occur more slowly than the millisecond responses in the synapse to the main excitatory neurotransmitter of the brain (glutamic acid) and the main inhibitory neurotransmitter (gamma amino butyric acid — aka GABA). Receptors on the presynaptic neuron for the transmitter it’s releasing are called autoreceptors, but the presynaptic terminal also contains receptors for other neurotransmitters.

Well at least, neurotransmitters aren’t released by the presynaptic neuron without an action potential which depolarizes the presynaptic terminal, or so we thought until [ Neuron vol. 82 pp. 63 - 70 '14 ]. The report involves a structure near and dear to the neurologist the striatum (caudate and putamen — which is striated because the myelinated axons of the internal capsule go through its anterior end giving it a striated appearance).

It is the death of the dopamine containing neurons in the substantial nigra which cause Parkinsonism. They project some of their axons to the striatum. The striatum gets input elsewhere (from the cortex using glutamic acid) and from neurons intrinsic to itself (some of which use acetyl choline as their neurotransmitter — these are called cholinergic interneurons).

The paper makes the claim that the dopamine neurons projecting to the striatum also contain the inhibitory neurotransmitter GABA.

The paper also says that the cholinergic interneurons cause release of GABA by the dopamine neurons — they bind to a type of acetyl choline receptor called nicotinic (similar but not identical to the nicotinic receptors which allow our muscles to contract) in the presynaptic terminals of the dopamine neurons of the substantial nigra residing in the striatum. Isn’t medicine and neuroanatomy a festival of terms? It’s why you need a good memory to survive medical school.

These used optogenetics (something I don’t have time to explain — but see http://en.wikipedia.org/wiki/Optogenetics ) to selectively stimulate the 1 – 2% of striatal neurons which use acetyl choline as a neurotransmitter. What they found was that only GABA (and not dopamine) was released by the dopamine neurons in response to stimulating this small subset of neurons. Even more amazing, the GABA release occurred without an action potential depolarizing the presynaptic terminal.

This literally stands everything I thought I knew about neurotransmission on its ear. How widespread this phenomenon actually is, isn’t known at this point. Clearly, the work needs to be replicated — extreme claims require extreme evidence.

Unfortunately I’ve never provided much background on neurotransmission for the hapless chemists and medicinal chemists reading this (if there are any), but medicinal chemists must at least have a smattering of knowledge about this, since neurotransmission is involved in how large classes of CNS active drugs work — antidepressants, antipsychotics, anticonvulsants, migraine therapy. There is some background on this here — http://luysii.wordpress.com/2010/08/29/some-basic-pharmacology-for-the-college-student/

The death of the synonymous codon – IV

The coding capacity of our genome continues to amaze. The redundancy of the genetic code has been put to yet another use. Depending on how much you know, skip the following three links and read on. Otherwise all the background to understand the following is in them.

http://luysii.wordpress.com/2011/05/03/the-death-of-the-synonymous-codon/

http://luysii.wordpress.com/2011/05/09/the-death-of-the-synonymous-codon-ii/

http://luysii.wordpress.com/2014/01/05/the-death-of-the-synonymous-codon-iii/

There really was no way around it. If you want to code for 20 different amino acids with only four choices at each position, two positions (4^2) won’t do. You need three positions, which gives you 64 possibilities (61 after the three stop codons are taken into account) and the redundancy that comes with it. The previous links show how the redundant codons for some amino acids aren’t redundant at all but used to code for the speed of translation, or for exonic splicing enhancers and inhibitors. Different codons for the same amino acid can produce wildly different effects leaving the amino acid sequence of a given protein alone.

If anything will figure out a way to use synonymous codons for its own ends, it’s cancer. [ Cell vol. 156 pp. 1129 - 1131, 1324 - 1335 '14 ] analyzed protein coding genes in cancer. Not just a few cases, but the parts of the genome coding for the exons of a mere 3,851 cases of cancer. In addition they did whole genome sequencing in 400 cases of 19 different tumor types.

There are genes which suppress cancer (which cancer often knocks out — such as the retinoblastoma or the ubiquitous p53), and genes which when mutated promote it (oncogenes like ras). They found a 1.3 fold enrichment of synonymous mutations in oncogenes (which would tend to activate them) than in the tumor suppressors. The synonymous mutations accounted for 20 – 40 % of somatic mutations found in cancer exomes.

Unfortunately, synonymous mutations have been used to estimate the background mutation frequency for evolutionary analysis, on the theory that they are neutral (e.g. because they don’t change protein structure, they are assumed not to change how the gene for the protein functions). Wrong. Wrong. They can change how much, or where, or what exons of a protein are included in the final product.

The prions within us

Head for the hills. All of us have prions within us sayeth [ Cell vol. 156 pp. 1127 - 1129, 1193 - 1206, 1206 - 1222 '14 ]. They are part of the innate immune system and help us fight infection. But aren’t all sorts of horrible disease (Bovine Spongiform Encephalopathy aka BSE, Jakob Creutzfeldt disease aka JC disease, Familial Fatal Insomnia etc. etc.) due to prions? Yes they are.

If you’re a bit shaky on just what a prion is see the previous post which should get you up to speed — https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/.

Initially there was an enormous amount of contention when Stanley Prusiner proposed that Jakob Creutzfeldt disease was due to a protein forming an unusual conformation, which made other copies of the same protein adopt it. It was heredity without DNA or RNA (although this was hotly contended at the time), but the evidence accumulating over the years has convinced pretty much everyone except Laura Manuelidis (about whom more later). It convinced the Nobel Prize committee at any rate.

JC disease is a rapidly progressive dementia which kills people within a year. Fortunately rare (attack rate 1 per million per year) it is due to misfolded protein called PrP (unfortunately initially called ‘the’ prion protein although we now know of many more). Trust me, the few cases I saw over the years were horrible. Despite decades of study, we have no idea what PrP does, and mice totally lacking a functional Prp gene are normal. It is found on the surface of neurons. Bovine Spongiform Encephalopathy was a real scare for a time, because it was feared that you could get it from eating meat from a cow which had it. Fortunately there have been under 200 cases, and none recently.

If you cut your teeth on the immune system being made of antibodies and white cells and little else, you’re seriously out of date. The innate immune system is really the front line against infection by viruses and bacteria, long before antibodies against them can be made. There are all sorts of receptors inside and outside the cell for chemicals found in bacteria and viruses but not in us. Once the receptors have found something suspicious inside the cell, a large protein aggregate forms which activates an enzyme called caspase1 which cleaves the precursor of a protein called interleukin 1Beta, which is then released from some immune cells (no one ever thought the immune system would be simple given all that it has to do). Interleukin1beta acts on all sorts of cells to cause inflammation.

There are different types of inflammasomes and the nomenclature of their components is maddening. Two of the sensors for bacterial products (AIM, NLRP2) induce a polymerization of an inflammasome adaptor protein called ASC producing a platform for the rest of the inflammasome, which contains other proteins bound to it, along with caspase1 whose binding to the other proteins activates it. (Terrible sentence, but things really are that complicated).

ASC, like most platform proteins (scaffold proteins), is made of many different modules. One module in particular is called pyrin (because one of the cardinal signs of inflammation is fever). Here’s where it gets really interesting — the human pyrin domain in ASC can replace the prion domain of the first yeast prion to be discovered (Sup35 aka [ PSI+ ] — see the above link if you don’t know what these are) and still have it function as a prion in yeast. Even more amazing, is the fact that the yeast prion domain can functionally replace ASC modules in our inflammasomes and have them work (read the references above if you don’t believe this — I agree that it’s paradigm destroying). Evidence for human prions just doesn’t get any better than this. Fortunately, our inflammasome prions are totally unrelated to PrP which can cause such havoc with the nervous system.

Historical note: Stanley Prusiner was a year behind me at Penn Med graduating in ’67. Even worse, he was a member of my med school fraternity (which was more a place to get a decent meal than a social organization). Although I doubtless ate lunch and dinner with him before marrying in my Junior year, I have absolutely no recollection of him. I do remember our class’s medical Nobel — Mike Brown. Had I gone to Yale med instead of Penn, Laura Manuelidis would have been my classmate. Small world

A primer on prions

Actually Kurt Vonnegut came up with the basic idea behind prions in his 1963 Novel “Cat’s Cradle”. Instead of proteins, it involved a form of water (Ice-9) which had never been seen before, but one which was solid at room temperature. Unfortunately, it also solidified all liquid water it came in contact with effectively ending life on earth.

Now for some history.

The first Xray crystallographic structures of proteins were incredibly seductive intellectually, much as false color functional magnetic resonance (fMRI) images are today. It was hard not to think of them as the structure of the protein.

Nowaday we know that lots of proteins have at least one intrinsically disordered (trans. unstructured) segment of 30 amino acids ore more. [ Nature vol. 411 pp. 151 - 153 '11 ] says 40%, and also that 25% of all human proteins are likely to be disordered (translation; unstructured) from end to end — basic on a bioinformatics program.

I’ve always been amazed that any protein has only a few shapes, purely on the basis of the chemistry — read this if you have the time — http://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/. Clearly the proteins making us up do have a relatively limited number of shapes (or we’d all be dead).

The possible universe of proteins from which our proteins are selected is enormously large. In fact the whole earth doesn’t have enough mass (even if it were made entirely of hydrogen, carbon, nitrogen, oxygen and sulfur) to make just one copy of the 20^100 possible proteins of length 100. For the calculation please see — http://luysii.wordpress.com/2009/12/20/how-many-proteins-can-be-made-using-the-entire-earth-mass-to-do-so/ — if you have the time.

So, even though it is meaningful question philosophically, just how common proteins with a few shapes are in this universe, we’ll never be able to carry out the experiment. Popper would say it’s a scientifically meaningless question, because it can’t be experimentally decided. Bertrand Russell would not.

Again, if you have time, take a look at http://luysii.wordpress.com/2010/08/08/a-chemical-gedanken-experiment/

Which, at long last, brings us to prions.

They were first discovered in yeast, and were extremely hard to figure out as they represented something in the cytoplasm which contained no DNA and yet which was heritable. The first prion was discovered nearly 50 years ago. It was called [PSI+] and it produced a lot of new proteins in yeast containing it (which is how its effects were measured) Mating [ PSI+ ] with [ psi-] (e.g. yeast cells without [ PSI+ ] converted the [ psi-] to [ PSI+ ]. It couldn’t be mapped to any known genetic element. Also [ PSI+ ] was lost at a higher rate than would be expected for a DNA mutation. The first clue that [ PSI+ ] was a protein was that it was lost faster when yeast were grown in the presence of protein denaturants (such as guanidine).

It turned out that [ PSI + ] was an aggregated form of the Sup35 protein, which basically functioned to suppress the ribosome from reading through the stop codon. If you need background on what was just said please see — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and the subsequent 4 posts. This is why [ PSI+ ] yeast produced longer proteins.Things began to get exciting when Sup35 was dissected so domains could be found which induced [ PSI+ ] formation. Amazingly these domains spontaneously formed visible fibers in vitro resembling amyloid in some respects (binding the dye Congo Red for one). Then they found that preformed fibers, greatly accelerated fiber formation by unpolymerized Sup35 — beginning to sound a bit lice Ice 9 doesn’t it. Yeasts have many other prions, but the best studied and most informative is the one formed from Sup35.

So that’s how prions were found (in yeast) and what they are — an aggregated form of a given protein in a slightly different shape, which can cause another molecule of the same protein to adopt the prion proteins new shape. Amazingly, we have prions within us. But that’s the subject of the next post.

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