COX-2 dependent regulation of mechanotransduction in human breast cancer cells- Part 2

It's been a few days, but now time to continue with what I started! (To recap, in an earlier post, I started looking at a paper about breast cancer metastasis called COX-2 dependent regulation of mechanotransduction in human breast cancer cells. I'm blogging about it in a desperate attempt to make reading journal articles more palatable.)

Results

The results section was actually right after the introduction in the paper, because I guess that's how most people read papers :P To my understanding, not many people look at the methods in great detail unless they are critiquing a paper (or blogging about it, I guess...?) which is probably why they were shoved in unceremoniously at the end, right before the references.

Anyway, results. Let's take a look.

Force-generating capacity of individual human breast cancer cells with different COX-2 expression and invasiveness

This was done using the Fourier transform traction microscopy (FTTM) that I talked about briefly in part 1. They put cells from 3 different cell lines (MCF-7, SUM-149 and MDA-MB-231) on an 8kPa elastic hydrogel coated with collagen. (8kPa was chosen because it's similar to the stiffness of breast tissue.) These cell lines differed in invasiveness: MCF-7 was the least invasive, followed by SUM-149 and finally MDA-MB-231.

When put onto the gel, these cells differed in several ways. Firstly, they differed in morphology (shape). MCF-7 cells were relatively rounded, SUM-149 was a bit more elongated, and MDA-MB-231 cells were quite a bit more elongated and polarised. (The picture in the paper looks somewhat dumbbell-shaped, with two rounder, slightly fatter ends with a straight bit connecting the two.) Cell size was also found to be significantly larger in MDA-MB-231 cells as compared to the other two, with a P value of <0.0001. (For those who don't know what a P value is, it's essentially a measure of how statistically significant a result is- i.e. how likely it is that you would have gotten that result due to chance alone. P values of <0.05 are generally considered to be statistically significant.) However, there were no significant differences between MCF-7 and SUM-149 cell sizes.

The different cell lines also exerted different amounts of traction on the gel. This was found by measuring the net contractile moment, which is a measure of contractile strength. Just like cell size, MCF-7 and SUM-149 did not differ significantly from each other, but MDA-MB-231 cells were significantly different, with a P value less than 0.01. MDA-MB-231 cells were found to have a contractile moment roughly 2.1 times that of the other two cells, and a contractile strength 2.9 times higher than the other cells.

Role for COX-2 in cell tractions?

To my understanding, this bit is all about using shRNA (short hairpin RNA) complementary to COX-2 mRNA, presumably to see how silencing COX-2 affects the ability of the cells to move around and do other stuff required to metastasise. Presumably shRNA silences expression of COX-2 mRNA by binding to it, preventing ribosomes and tRNA from translating it. Anyway they found that MDA-MB-231 cells expressing the shRNA were relatively more packed with their neighbouring cells, moved more slowly and underwent less cytoskeleton remodelling, probably because there were also decreases in some of the gene transcripts involved in cytoskeleton remodelling. I can't really comment on their data, though, because it was all in supplementary images which don't seem to be attached to the paper. (And yes, I did download the "Supplemental" file, but I only got the videos, which don't really show a lot. I assume they're meant to show the different amounts of movement exhibited by cells with and without the shRNA, but it doesn't say which one's which.) Traction in the shRNA cells tended to be less than that of normal MDA-MB-231 cells, but this wasn't statistically significant. Or so they say. They didn't show their data, and I mean they literally wrote "(data not shown)." Not very helpful. (That can be my #1 critique of the paper. Mainly because I don't know enough to critique anything else.)

They were kind enough to provide their graphs for the next bit, though. Their next bit was essentially just putting individual regular MDA-MB-231 cells and individual MDA-MB-231 cells with shRNA onto the 8kPa matrix and seeing how they responded. I guess their mini-hypothesis here would be that the shRNA cells wouldn't spread as much or generate as much force as the normal ones. In fact, that's what happened: the COX-2-silenced cells had a roughly 35% reduction in cell spreading and a roughly 60% reduction in net contractile moment. They provided some graphs too, which was really nice of them.

Aside from just testing this with 8kPa matrix, they also tested the COX-2-silenced and normal cells on matrices of different stiffnesses: 1kPa, 8kPa and 20kPa, to be exact. COX-2-silenced cells showed reduced cell spreading and net contractile moment across all matrix stiffnesses. Another thing that was tested was expression of the mechanosensitive integrin proteins beta 1 and beta 3. The COX-2 silenced cells showed decreased expression of both of these proteins compared to normal cells (P values for beta 1 were 0.035, <0.001 and 0.003 on 1kPa, 8kPa and 30kPa, respectively; P-values for beta 3 were <0.001, 0.035 and 0.008). Weirdly enough though, they didn't talk about this in the results: instead they said that the regular MDA-MB-231 cells showed increased expression of beta 1 (but not beta 3) with increased matrix rigidity. I'm not convinced of this looking at their results, because their graphs seem to show that beta 1 expression decreased on the 30kPa as compared to the 8kPa. They then went on to say that this effect that they apparently saw in the normal cells wasn't seen in the COX-2-silenced cells, which I guess I agree with.

Propagation of cellular traction is mediated by a feed-forward mechanism involving COX-2-PGE₂ axis

Another interesting finding from plonking cells on matrices of different stiffnesses is that regular MDA-MB-231 cells appear to increase their production of PGE₂ at increasing stiffnesses. COX-2-silenced cells, on the other hand, had much lower levels of PGE₂ that did not increase with stiffness. The next step was to see whether COX-2 had its effects by increasing PGE₂ levels, or through other means. This was tested by adding exogenous (from outside) PGE₂. Adding PGE₂ increased net contractile moments for both regular cells and COX-2-silenced cells. According to their figure legend, however, there were decreases once more than 100nM PGE₂ was added, but they somewhat rudely left them off the graph.

They also tested out the cytoskeletal stiffnesses (or at least I think that's what they tested, but I could be 100% wrong) using Magnetic Twisting Cytometry, which I also mentioned in part 1. Sadly their data is hidden away in mysterious Supplementary Figure land, and then there's some random note about PGE₂ and the stiffness of human airway smooth muscle cells, which is either irrelevant or some serious typo.

Finally, they also tested out the effects of exogenous PGE₂ on the cytoskeletal stiffnesses of all three cancer cell lines that I mentioned earlier (MCF-7, SUM-149 and MDA-MB-231, from least metastatic to most metastatic). Cytoskeletal stiffnesses were again measured using Magnetic Twisting Cytometry. Stiffness was found to increase with the addition of PGE₂ for SUM-149 and MDA-MB-231 cells, but not for MCF-7 cells.

I'll discuss the discussion (um... yeah) in a later post. But there'll probably a bit of time between this post and the next on this topic, because I want to revise some respiratory stuff first. (Flow-volume loops, I'm looking at you.) And then there's also anatomy... and biochemistry... and pharmacology... and all of the cardiovascular physiology stuff that I still haven't written about... So much to learn, so little time!

COX-2 dependent regulation of mechanotransduction in human breast cancer cells- Part 1

Long story short I have to do a research placement this semester, which involves trying to read journal articles. Emphasis on the "trying" because those buggers are a pain. I celebrate when I can work out what the hell is going on. Blogging about articles is my desperate attempt to make reading about them less painful.

Anyhow, the article that I've picked is called COX-2 dependent regulation of mechanotransduction in human breast cancer cells, by A-Rum Yoon, Ioannis Stasinopoulos, Jae Hun Kim, Hwan Mee Yong, Onur Kilic, Denis Wirtz, Zaver M Bhujwalla and Steven S An. I'm not going to read the abstract just yet because some random on Facebook said that sometimes reading the abstract can bias the way you read the rest of the article. Of course, you shouldn't just go around believing everything randoms on Facebook say, but I don't see the harm in trying this.

On to the introduction...

Introduction

Breast cancer sucks. Metastasis doubly sucks because then it's like two cancers for the price of one, which is a deal that you wouldn't want to take up, but it's not like you have any choice in the matter because not enough is known about how and why cancers metastasise. This is why we do research!

This article's going to focus on the metastasising part, so let's sum up very briefly what we know about it, or at least what we think we know about it. Essentially some traits accumulate, allowing cells to escape, travel through the extracellular matrix (ECM) and enter the systemic circulation. The cells then travel to distant organs, where they burrow their way into the ECM and set up camp in a new tumour. Also according to this introduction inflammation is currently assumed to play a part in all of these processes, but there's still a lot of fuzziness and grey area to explore here.

This is where COX-2 comes in! COX-2 stands for "cyclooxygenase-2," which I mentioned before when I wrote about structure-activity relationships and the coxibs. COX-2 mediates inflammation and converts arachidonic acid into biologically active lipids known as prostaglandins (notably prostaglandin E2, or PGE₂). The interesting part is that COX-2 has been found to be expressed at higher levels in malignancies, and PGE₂ has also been implicated in metastasis. Previous studies have also shown that inhibition of COX-2 synthesis by RNA interference (a process in which RNA molecules inhibit the gene expression, by destroying the mRNA or otherwise) reduce the expression of ECM metalloproteinases (metalloproteinases are protein-destroying enzymes that require metal to work properly; those in the matrix have been related to invasion of a cancer). This, in turn, was shown to decrease the ability of metastasising cells to invade a new tissue.

Well that's all interesting, but what's the actual aim of this study? To my understanding, the aim of the study was to find out whether COX-2 and mechanotransduction (sensing an external force and turning it into some kind of response from the cell) are related, but I could be wrong: I didn't find it particularly clear what the actual aim was from the introduction alone. But maybe I'm just dumb. Idk.

Methods

The "methods" section was stuck right at the end, because let's face it, unless you're trying to critique a paper or whatever that tends to be the first part you're going to skip. But I'm actually trying to go over the paper here, so let's have a look at what's here.

Cell lines and culture materials

This section just listed off the ingredients of the cell media, as well as the temperature and percentage of CO₂. Not sure if that's particularly relevant to me unless I want to replicate the experiment or double check that they used the right media for the cells (not that I know anyway :P).

Preparation of elastic matrix

As with many papers involving mechanotransduction, they've made an elastic polyacrylamide hydrogel out of acrylamide and bisacrylamide. Acrylamide basically makes long chains, whereas bisacrylamide crosslinks these chains. The more crosslinks there are (i.e. a higher percentage of bisacrylamide), the more rigid the gel will be. They made two similar mixtures: one without any extra stuff added, and one with fluorescent microbeads added so that traction force microscopy could be done: more on this in a bit.

Unfortunately, cells themselves can't adhere directly to the matrix, which is why they also coated both gel blocks with type I collagen. Type I collagen can't adhere directly to the matrix either, though, so a cross-linking particle called sulfo-sanpah was used.

Fourier transform traction microscopy (FTTM)

From my understanding, this procedure was used to quantify the contractile stress between the cell and synthetic ECM (i.e. the elastic matrix and other stuff on top). This used the gel with the fluorescent microbeads. Essentially a cell was put onto the gel block and allowed to stabilise. Pictures of the fluorescent microbeads were taken at various times. Afterwards, they used trypsin to detach the cells and then took another picture as the reference. The distance the beads move can be related to the force that the cells exert on the matrix. I don't really know what Fourier transform traction cytometry is about, and finding out seems to involve reading another paper, and this just reeks of looking up words in my Chinese dictionary only to have to look up definitions for words in the definition and so forth.

Magnetic twisting cytometry (MTC)

Okay, I've never heard of this one before, plus I've never done physics, so here's my really basic understanding of how it works. Essentially they get a magnetic bead and anchor it to the cytoskeleton through cell surface integrin receptors and the tripeptide RGD (arginine-glycine-aspartic acid). This bead is magnetised and twisted. As it twists, the cell resists. A camera can be used to detect the lateral bead displacements, and algorithms used to work out the stiffness of the cells. Or something.

Detection of COX-2 activity

Enzyme immunoassays (EIA) were used to detect the concentration of PGE₂, which in turn was used to assess COX-2 activity (remember, COX-2 converts arachidonic acid into PGE₂). I feel that I don't know as much about how EIA works as I should, but thankfully I found a handy video:

In the case of this paper, they appear to have quantified the amount of binding by using a spectrophotometer, which quantifies the intensity of the signal (i.e. how much the colour changed). They would have presumably also used a standard curve, which takes samples that have the substance at known concentrations, for comparison. (Or at least I'm just assuming that because that's what we've done in labs at uni.)

Fluorescence-activated cell-sorting (FACS) analysis

This was used to quantify the cell surface expression levels of beta 1 and beta 3 integrins. Essentially some cells were divided into two groups and incubated with specific FITC-conjugated antibodies. One group received antibodies against integrin beta 1, whereas the other received antibodies against integrin beta 3. FITC is a green fluorescent dye. Its fluorescence can be detected and quantified by a machine known as a flow cytometer.

Statistical analysis

The main statistical tests they used were Student's t-test and ANOVA (Analysis of Variance). I don't know much about either of these tests to be honest, and after typing up all that stuff above, I don't really feel like looking them up right now either. These can potentially be a topic for a later post.

Ligand gated ion channels

In my last pharmacology post, I spoke about the four main classes of receptors: ion channels, G-protein coupled, enzyme-linked and nuclear/DNA-linked. In these next few posts, I'll be expanding on each of these four categories.

Provide examples of ligand-gated ion channels.

My previous post already provided one: nicotinic ACh receptors, which are also Na⁺ channels. (These also got a mention in my post on the autonomic nervous system. If you're wondering about muscarinic receptors, they're actually G-protein coupled.)

A second example of a ligand-gated ion channel is GABA_A receptors. (GABA_C receptors do the same thing, but we're just going to ignore them for now. Oh, and there's also GABA_B receptors, but they're G-protein coupled.) GABA receptors are Cl^- channels which hyperpolarise the cell when open.

Both of the above receptors (nicotinic and GABA_A) have similar structures. I'll discuss the structures in a bit...

Be able to describe the basic structure and transduction mechanisms of ligand-gated ion channels. 

Ligand-gated ion channels usually have around 4-5 subunits that make up the "channel." These subunits are imaginatively called alpha, beta, gamma, delta etc. Each subunit has both the N- and C-terminals located extracellularly, with four alpha helices crossing the membrane. In the case of the two previous receptors mentioned (nicotinic and GABA_A), there are five subunits present: alpha, alpha, beta, gamma and delta. Each alpha subunit must have a molecule of agonist bound for the subunits to turn slightly, opening the channel and allowing ions to pass through.

The movement of ions through the membrane may make the inside of the cell more positive (depolarisation) or more negative (hyperpolarisation). The cell is usually negatively charged compared to its environment, with a "resting potential" of around -70mV. In some cells (particularly neurons and skeletal muscle), once the cell reaches around -50mV, an "action potential" (i.e. a rapid shift from negative to positive charge inside the cell) is initiated. This results in neurons firing, skeletal muscle contraction, and so on.

Another interesting point to mention is that the lining of the channel (i.e. the amino acid groups) might influence which ions pass through a channel once it opens. For positively-charged ions such as Na⁺, the lining will have more negatively-charged amino acids such as aspartic acid and glutamic acid, whereas the opposite will be true for negatively-charged ions.

Be able to explain how interaction of a ligand with an ion channel is coupled to a biological effect within the cell. 

I feel like I've already explained this in my last few paragraphs, so I'm going to expand on this by talking about antagonists and long-acting agonists.

As mentioned in my previous post, pancuronium is a competitive antagonist of the nicotinic ACh receptor (i.e. it binds to the receptor without having an effect, also preventing ACh from binding and having an effect). When the ACh receptor is blocked, the Na+ channel is prevented from opening. This prevents depolarisation of the cell, which stops skeletal muscle from having an action potential and contracting. Hence, pancuronium is used as a local anaesthetic as it stops muscles from twitching during surgery.

Another kind of drug is a long-acting agonist. An example of a long-acting agonist is suxamethonium. Long-acting agonists also bind to the receptor, but they keep the channel open. This prevents the cell from depolarising over and over again. Since such repetitive depolarisations are necessary for sustained contraction, suxamethonium is good for short-term muscle relaxation in short surgical procedures.

Autonomic Nervous System

There's just one unit that I've been neglecting to blog about, and that's Physiology! I blame the lack of tests for me to stress about... not like I'm complaining ;) (Also the main reason why I'm blogging about Physiology and not finishing up those last few posts about Anatomy is because there are a few things that I want to clear up before I write my next post.)

These first few posts are going to be about the autonomic nervous system and the endocrine (hormonal) system, as they help to regulate many functions in the body (and physiology is all about how things function). In doing so, they maintain "homeostasis," or a stable environment in the body.

These control systems can be spoken about has having sensors, integrators and effectors: the sensor is the bit that notices if something is a bit out of line, the integrator figures out what to do, and the effector gets a response done. For example, an increase in blood pressure is sensed by baroreceptors (pressure receptors) located in the carotid sinus and aortic arch. These send signals to the cardiovascular control centre located in the medulla in the brain, which in turn sends out signals to speed up or slow down the heart, returning blood pressure to normal. The sensors here are the baroreceptors, the integrator is the medulla and the effectors are the nerves going to the heart.

About Nerves...

Nerves can be categorised depending on their direction. Afferent nerves go towards the CNS, whereas efferent nerves go away from the CNS (think of the brain telling efferent nerves to "eff off"). Afferent nerves include sensory nerves, whereas efferent nerves include the somatic and autonomic nervous system. The somatic nervous system covers most stuff that we think of as voluntary, whereas the autonomic nervous system deals with involuntary stuff like heart rate and breathing, and is thus very important for keeping lots of things in our body under control.

A very quick recap of what a neuron is: a neuron is, in short, a nerve cell that "sends messages." It has a cell body, which is where most of its organelles are. It is surrounded by dendrites, which pick up messages from other neurons. Neurons also have a long thin axon through which impulses travel. When the impulse reaches the end, neurotransmitters are released into the gap between the end of one neuron and the dendrites of the next (this gap is also known as a synapse). Many autonomic neurotransmitters, however, are actually released from varicosities (irregular expansions on the axon), which allows for diffuse, non-directional release.

In the autonomic nervous system, the CNS sends out one nerve, which synapses with another, which synapses with the target organ. The bit where autonomic nervous system (hereafter referred to as ANS because I can't be bothered typing it out in full any more) neurons synapse is also known as a ganglia. The first neuron is known as a preganglionic neuron, whereas the neuron afterwards is known as the postganglionic neuron. It sounds somewhat inefficient having two nerves to go to one organ, but in fact this system has advantages as multiple neurons can stimulate one neuron, or vice versa: one neuron can stimulate multiple other neurons.

The autonomic nervous system can also be further categorised into three different divisions: sympathetic, parasympathetic and enteric (gut). I'm only going to cover the first two. They have distinct anatomical and physiological differences. Most organs receive innervation from both systems, though there are some exceptions (for example most blood vessels only receive sympathetic innervation). The two systems also tend to have opposite effects, but not always: both stimulate the secretion of saliva, but the contents of saliva may be different depending on which system has stimulated the secretion. Another common misconception is that only one of these systems is active at a time: in reality, usually both systems are active, though one may predominate depending on what's going on.

Sympathetic Nervous System

Sympathetic nervous system nerves leave from the thoracic and lumbar regions of the spinal cord as white rami (see my anatomy posts for more information). Most of the ganglia lie in a long line known as the sympathetic trunk, located close to the spinal cord. The preganglionic neurons tend to be short, as the sympathetic trunk is not too far away, but the postganglionic neurons are long. The fact that the synapses are pretty much all in the same place may also be the reason why the sympathetic nervous system tends to act pretty much as one unit, but I'm not 100% sure about this.

The sympathetic nervous system is well-known for participating in the "fight-or-flight" response, though it's also pretty important in more mundane things such as making sure that our blood pressure doesn't drop when we get out of bed in the morning. It causes an increased heart rate and cardiac output (which is heart rate multiplied by stroke volume- more on this when I write about the heart later on). It also causes vaso- and venoconstriction (i.e. constriction of arteries and veins). Other effects include the dilation of pupils, opening of respiratory airways, breakdown of glycogen and fat stores to provide energy, increased sweating, redistribution of blood flow away from the digestive and urinary systems and towards the muscles and activation of the adrenal medulla which synthesises hormones which compound the effects of the sympathetic nervous system.

About the adrenal medulla: the medulla is the middle part of the adrenal glands, which are located on top of the kidneys. The adrenal medulla secretes catecholamines, of which there are two main ones you need to know: adrenaline and noradrenaline (a.k.a. epinephrine and norepinephrine). These are released by chromaffin cells, which are stimulated directly by preganglionic neurons in the sympathetic nervous system (there is no postganglionic neuron required here- the chromaffin cell pretty much is the postganglionic cell). Adrenaline and noradrenaline pretty much do the same stuff that the rest of the sympathetic nervous system does (in fact, noradrenaline is a common neurotransmitter used in the sympathetic nervous system, as I'll expand on in a bit).

Now time to talk about neurotransmitters and receptors! All autonomic nervous system preganglionic nerves secrete acetylcholine (ACh), regardless of whether they are part of the sympathetic or parasympathetic nervous system. AcetylCHOLINE is recognised by CHOLINErgic receptors, of which there are two main ones: nicotinic and muscarinic. Pretty much the only ones involved in the sympathetic nervous system are nicotinic receptors, as they're found on postganglionic cell bodies as well as the postsynaptic membranes of skeletal muscle cells. After ACh has done is job, it is rapidly broken down by extracellular acetylcholinesterases.

Postganglionic cells secrete noradrenaline- see, I told you it was commonly used in the sympathetic nervous system! The only exception is sweat glands- the postganglionic cells that innervate sweat glands are also cholinergic. (Chromaffin cells might also be considered an exception as they don't have postganglionic neurons: instead, they are innervated directly by preganglionic cholinergic neurons.) Noradrenaline binds to adrenergic receptors, which also bind adrenaline. There are two types of adrenergic receptors, each with two subtypes. Here are examples of where they are located and what effects they have (bear in mind that this is as far from a conclusive list as you could possibly get- well, without leaving the page blank, of course).

• alpha 1 receptors are located in the smooth muscle of blood vessels, where they stimulate them to contract.
• alpha 2 receptors are located in the pancreas, where they inhibit insulin release.
• beta 1 receptors are located in the heart, where they stimulate the heart to increase the rate and force of contraction. These can be blocked by the drug propranolol.
• beta 2 receptors are located in the smooth muscle of airways, where they have an inhibitory effect, resulting in relaxation. They have a greater affinity for adrenaline than noradrenaline (all other subtypes have roughly equal affinity for both adrenaline and noradrenaline).

Another important thing to remember is that specific neurotransmitters cannot said to have a certain effect. It's how the signal is transduced by the cell that matters. Here's another way of explaining that that might make more sense: you can't say that "noradrenaline stimulates stuff" because, as I just listed above, it can cause contraction or relaxation, depending on the receptor and target cell.

Before I move on, just a quick word on how the body gets rid of acetylcholine and noradrenaline. They can be taken back up by the sympathetic nerves, where they can be broken down by monoamine oxidase (so I wonder what happens in patients who take MAOIs- monoamine oxidase inhibitors? Hmm...). They can also be inactivated by catechol-O-methyltransferase in the liver.

Parasympathetic Nervous System

The parasympathetic nervous system generally has opposing effects to the sympathetic nervous system. While the former is all about "fight or flight," this system is all about "rest and digest."

Anatomically they are fairly different. Parasympathetic nerve fibres arise from the base of the skull or the sacral region of the spinal cord. Preganglionic neurons don't synapse until they are quite close to their target organs. This is probably why parasympathetic stimulation tends to be more specific.

As I mentioned, all autonomic preganglionic neurons release ACh. Parasympathetic postganglionic neurons also release ACh. Target cells in the parasympathetic system have muscarinic receptors, rather than the nicotinic ones found nearly everywhere else. Muscarinic receptors can be blocked by the drug atropine.

Autonomic Conflict

As I've alluded to previously, the idea that only one of these two systems is active at a time is a myth. In fact, in some cases, both systems can be stimulated simultaneously. An example of this is the dive reflex. When submerged in cold water, the parasympathetic nervous system is activated, slowing down the heart rate so as to conserve oxygen. At the same time, the sympathetic nervous system is activated due to stimulation by temperature receptors. Some people can get arrhythmias (abnormal heart rhythms) by doing this, though, so do be careful if you want to test this. (Oh, and since I've warned you, if you die it's not my fault.)

Mechanics of Breathing: Structure and Function of the Thoracic Wall

Now we're onto the thorax stuff! These next few lectures were incredibly dense, so much so that one of them had extra slides tacked on the end for us to read in our own time >_>

Anatomy of the thoracic wall- bones, joints, muscles

The thoracic wall is made up of the rib cage, with its 12 ribs and sternum, as well as the gaps between ribs, the parietal pleura (the layer of the lungs that adheres onto the ribs- more on this later) and the muscles and bones of the upper limb.

Now I'm going to go into each of these elements in more detail, because as you've probably realised by now, anatomy seems to be a lot about dissecting the crap out of stuff and finding out as many details as you can before your head explodes. Anyway...

So back on the topic of the rib cage. There are 12 pairs of ribs which all attach to vertebrae, but not all attach to the sternum. The first seven ribs attach directly onto the sternum and are sometimes known as "true ribs" (well, there's some debate over whether the 7th rib counts as a true rib, but there you go). Ribs 8-10 attach to the cartilage of rib 7 and are sometimes known as "false ribs." The last two ribs, 11 and 12, do not attach to the sternum at all and are thus called "floating ribs."

The ribs also differ in their attachments to the vertebrae. Ribs have a head, a tubercle (which is a little bump near the head) and a body that ends in cartilage. The head articulates with facets on the body of the vertebrae. Most articulate with demifacets on two adjacent vertebrae as well as the disc in between them; however, ribs 1, 11 and 12 articulate with a single facet on T1, T11 and T12, respectively. The tubercle articulates with facets on the transverse processes (ribs 11 and 12 lack this joint). These joints are known as costotransverse joints (costo = rib), and are shaped differently as you go down the spine. The upper ribs tend to have more rounded costotransverse joints, whereas the lower ribs tend to have flatter surfaces at the joints. This also has implications in how they move: upper ribs tend to move up and forward in a "pump handle" motion, increasing the anterior-posterior diameter; lower ribs tend to move up and out in a "bucket handle" motion which increases the transverse diameter.

As mentioned before, ribs 1-7 attach to the sternum. The sternum itself can be broken down into various sections. The very top "notch" is known as the sternum notch (creative, I know). The top bony bit is called the manubrium, and it joins onto the body of the sternum at an angle known as the sternal angle (also very creative). This sternal angle is in line with T4. (Not sure what the significance of this is, but they keep drilling it in, so I assume it's important.) The very bottom part of the sternum is known as the xiphoid process, or xiphisternum.

Believe it or not, there are joints within the sternum. The joint between the manubrium and the body is a symphysis; however, partial ossification does occur. There is also a symphysis between the body and the xiphisternum, but this one might not ossify, which is why the xiphoid processes have fallen off some cadavers. There are also joints between the sternum and the ribs. Between the cartilage of the ribs and the sternum, there are small joints with cavities that are kinda like synovial joints. There are similar joints between the cartilages of the false ribs. Between ribs and cartilage, however, the matrix of the bone and cartilage just runs continuously into each other.

Now time to go into the muscles in a bit more detail! The true thoracic wall muscles are the external intercostals, internal intercostals and the inner layer of muscles, which consists of the transversus thoracis, innermost intercostals and subcostals. These all run between the ribs.

The fibres of the external intercostals run from top left to bottom right. When they contract, they lift the ribcage up and out. They are the second most important muscles in inspiration (the diaphragm is the most important muscle). The internal intercostals are the opposite: they run from bottom left to top right, and can pull the ribs down and inwards during forced expiration. Intercostal veins, arteries and nerves lie between the internal intercostals and the innermost layer. They are actually in that order (vein, artery, nerve) from top to bottom, and can be remembered by the acronym VAN.

Finally I'll just talk a bit about the diaphragm, because it is the divider between the thoracic and abdominal cavities. Also, it's important in breathing. The central tendon of the diaphragm is located in the centre. Around the edges, the diaphragm is attached to stuff. It is attached to the xiphisternum, ribs 7-12 and the lumbar vertebrae. At the point of attachment with the lumbar vertebrae, the diaphragm has two "legs," or crura. The left crus attaches to L1 and L2, whereas the right crus attaches to L1, L2 and L3. (Just in case you wanted more random details to remember!)

Lungs and pleural cavity

The lungs are covered by pleural membranes which surround a pleural cavity filled with a thin layer of fluid. This cavity originally developed from the coelomic cavity. The part of the pleural membrane that adheres to the lung is known as the visceral pleura, whereas the part of the pleural membrane that adheres to the chest wall is known as the parietal pleura. The pressure within the pleural cavity is lower than that of atmospheric pressure (around 756 mmHg as opposed to 760 mmHg) so sometimes it is said to have "negative pressure." This "negative pressure" also "sucks" the lungs towards the chest wall, thus expanding the lungs. If the pleural membranes become ruptured and the pressure becomes equal to that of atmospheric, the lungs will collapse. This is known as a pneumothorax.

Movements and their role in breathing

Breathing is all about changing the size of the thoracic cavity. Boyle's law states that pressure and volume are inversely proportional: as volume increases, pressure decreases and vice versa. Also, gases tend to move from an area of higher pressure to an area of lower pressure. Hence increasing the size of the thoracic cavity will decrease the pressure relative to atmospheric pressure, inducing air to "rush in." The opposite is true for decreasing the size of the thoracic cavity.

In inspiration (i.e. breathing in), the diaphragm and external intercostals are of utmost importance. The diaphragm moves downward in order to increase the size of the thoracic cavity. In doing so, the abdominal organs are compressed (this is why your belly moves out when you breathe in). When the abdominal organs cannot be compressed any further, the ribs are elevated. The balance between the use of the diaphragm (so-called "abdominal breathing") and external intercostals (thoracic breathing) can change depending on many factors, such as posture. In forced inspiration other muscles, such as the scalenes and sternocleidomastoid, may also be used to help lift the ribs.

No muscular effort is required during quiet expiration: instead, the muscles involved in inspiration simply relax. In forced expiration, however, other muscles such as the rectus abdominus (colloquially known as "abs") can help out.

Whew! That was long...

The Articular System

I've spoken a fair bit about the specifics of the vertebral column, but it's time to go a bit more general and talk about the joints that are found between bones all over the body. Yay!

Types of Joints

Joints can be classified in different ways. They can be classified in terms of moveability (i.e. moveable vs. immoveable) or via their structure (fibrous, cartilaginous or synovial). These are probably best represented in a table. Good thing I had one prepared earlier (for my end-of-topic revision thing, hehe).

I don't think there's much else that I can add to the above diagram, other than a few more random factoids about each joint type. (I'll only cover the fibrous and cartilaginous joints, as I'm going to ramble on about synovial in a bit.)

• Suture
• Fibrous tissue used: dense regular connective tissue
• Examples: Coronal suture, frontal suture etc. (basically sutures in the skull)
• Tend to fuse with age
• Syndesmosis
• Fibrous tissue used: interosseous ligaments, which are made of dense regular connective tissue
• Examples: radioulnar syndesmosis, tibiofibular syndesmosis
• Synchondrosis
• Examples: epiphyseal growth plates (in children), between the first rib and manubrium. (Synostosis is the fusion of two bones, such as when the epiphyseal plate closes in children.)
• Symphysis
• Movement varies according to thickness
• Cartilage used: articular hyaline cartilage (capping the bones) and fibrocartilage (between bones)
• Examples: pubic symphysis, intervertebral discs, between sacral and coccygeal bones

Synovial Joints

Synovial joints are kind of special as they have both fibrous and cartilaginous bits. Articular hyaline cartilage covers the surfaces of bones involved in synovial joints while the entire thing is enclosed in a fibrous capsule. The inside of the capsule is lined with a synovial membrane that produces synovial fluid, which lubricates and nourishes the joint.

As mentioned in the picture above, cartilaginous bits (i.e. articular cartilage and menisci- more on menisci later) get no nerve supply. The joint capsule, ligaments and tendons do, however. There's also a neat little law called Hilton's Law that states that if a nerve supplies a muscle that crosses a joint, it will also supply sensory fibres to that joint.

The blood supply is a little different. Bones and muscles have their own blood supply. The synovial membrane is supplied by small arteries, and the fluid it produces nourishes the articular cartilage. These arteries also have many anastomoses (i.e. alternative routes for blood) just in case some of the arteries are squashed due to the positioning of the joint. Fibrous and cartilaginous parts do not require a rich blood supply.

Special Features of Synovial Joints

Not all synovial joints are created equally. Some have special features which make them more suitable for various purposes. Let's take a look at some of these special features:

• Labria- these are like "lips" that deepen the socket of ball and socket joints (probably allowing for greater stability?)
• Bursae- these are like synovial joints without the articular cartilage. These normally form between tendons and bones.
• Intra-articular discs- these are fibrocartilaginous discs that divide the joint cavity in half. Usually different movements occur on different sides of the disc. The temporomandibular joint (between the temporal bone and the mandible) is one example of a joint with an intra-articular disc. Above the disc, protraction and retraction occur; below the disc, elevation and depression occur.
• Menisci- these are like partial intra-articular discs. There are different movements above and below these as well.
• Fat pads- these are like "soft menisci." They are more likely to be found in facet joints.

Movement of Synovial Joints

The movement of synovial joints depends a lot on the shape of the joint and the bones that form it.

Uniaxial joints have one degree of freedom- for example, they can usually flex/extend, or rotate, but not both. These tend to be bicondylar in shape (i.e. have two little "bumps" or condyles), or pivot joints.

Biaxial joints have two degrees of freedom, usually flexion/extension and abduction/adduction. These tend to be condylar or saddle joints.

Finally, multiaxial joints can flex/extend, abduct/adduct and rotate. These include ball and socket joints as well as plane/facet joints.

Contents of the Vertebral Canal

After a long break from blogging about anatomy, I figured that I should probably start up again. I'm going to pick up from where I left off last time: talking about the vertebral column. This time, I'm going to talk about the contents of the vertebral canal (basically the space in which the spinal cord etc. sits).

The Vertebral Canal

The vertebral canal, as I just said, is the space in which the spinal cord sits. It's also essentially the "tunnel" made up out of the vertebral foraminae. (Okay, that was two sentences with a whole lot of nothing. I'm sorry.)

The anterior part of the vertebral canal (i.e. what you see if you're sitting in the canal and looking forward) is made up of bodies, discs, and the posterior vertebral ligament (the ligament that runs along the backs of the vertebral bodies- see my post about intrinsic postvertebral muscles).

The posterior side is made up of the laminae, the ligamentum flavum ("yellow" ligament connecting laminae of adjacent vertebrae) and the zygapophyseal joints (a.k.a. Z joints for those of you who can't spell- or be bothered to spell- zygapophyseal). The posterior wall is deficient in the lower part of the sacrum. This part is known as the sacral hiatus.

Finally, the lateral sides of the canal are simply made of intervertebral foraminae (i.e. holes between vertebrae) and the pedicles (which are the bony bits separating foraminae).

The canal is not the same size all the way down. It is larger where the spinal cord is larger (presumably due to plexuses- clusters of nerves- or something?). It is also larger where there is more potential for movement.

Meninges

There are three meninges, which are layers that cover the spinal cord and brain. They are the dura mater, arachnoid mater and pia mater (from outside in). The arachnoid and pia are known collectively as leptomeninges, and are derived from neural crest ectoderm.

The outermost layer is the dura mater. It continues on from the dura mater of the brain, attaching to the circumference of the foramen magnum on its way down. Most of its other attachments are anterior: to the posterior side of the bodies of C2 and C3 and loosely to other areas of the posterior longitudinal ligament. It does, however, also attach to the dorsal side of the coccyx. The dura has "sleeves" which protect nerves as they leave through the intervertebral foraminae.

Although the dura does have some attachments with the posterior longitudinal ligament and whatnot, there is an important space between the dura mater and the inside of the vertebral canal. This space is known as the epidural space, and it's where fat and the internal vertebral venous plexus (basically a crapload of veins) can be found. It also extends a short way into the intervertebral foraminae. The significance of this epidural space is that drugs and so forth injected in this space can essentially traverse the vertebral column.

Another space worth mentioning is the subdural space. The subdural space is between the dura and the arachnoid, and normally doesn't exist because the arachnoid is pressed up against it due to the pressure of CSF (cerebrospinal fluid).

Now on to talking about the leptomeninges (pia and arachnoid mater)! The leptomeninges begin as a single membrane and are separated when CSF begins to flow and push them apart. They don't separate apart that cleanly, however, which is why there are random strands of tissue crossing the gap. The space between them is known as the subarachnoid space, and it's where CSF flows through the canal. The pia adheres closely to the spinal cord, but it also has projections called denticulate ligaments which pierce the arachnoid and attach to the inside of the dura, aiding stability of the spinal cord within the canal. These ligaments continue down to T12.

The subarachnoid space (which, as I just said, is between the arachnoid and pia), continues as far as the dorsal root ganglion. Past here, to my understanding the meninges essentially become continuous with the coverings of the peripheral nerves. From out to in, these are the epineurium, perineurium and endoneurium.

Spinal Cord

Unlike in embryos, the spinal cord in adult humans does not continue down the entire length of the spine. This is because the spinal cord stops growing before the spine does. The spinal cord ends at L1, and this ending is known as the conus medullaris. The conus medullaris is attached to the base of the spine by a thread of fibrous tissue known as the filum terminale. Another important structure here is the cauda equina- these are all of the nerves that are travelling downwards to exit the spine lower down (i.e. through L2-L5 or the sacral region).

The subarachnoid space continues further than the spinal cord. It continues all the way down to S1/S2, which is handy for doing a lumbar puncture: lumbar punctures are usually done between L3 and L4 or between L4 and L5, where the subarachnoid can be accessed without fear of accidentally puncturing the spinal cord. Below this level, the meninges continue and cover the filum terminale. The epidural space, with its fat and veins, is still around too.

Now for a bit more information about the spinal cord itself! Like the brain, the spinal cord has white matter (myelinated axons) and grey matter (nerve cell bodies). However, unlike the brain, the grey matter is on the inside whereas the white matter is on the outside. The closer up the spinal cord you go, there more white matter there is. More grey matter can be found where there are bigger nerves, for example near the brachial plexus (cervical enlargement, C5-T1) and lumbosacral nerves (lumbar enlargement, L2-S2).

Another important point to remember is that sympathetic nerves can be found around T1-L2 and parasympathetic nerves can be found around S2-4 (the pelvic splanchnic nerves). These areas have lateral horns of grey matter in addition to the ventral and dorsal horns found throughout the spinal cord.

You might have noticed that I haven't given C1-C5 much love. Well, they do have their own specialisations too. They give rise to the spinal accessory nerve, which innervates the sternomastoid and trapezius muscles. The roots of the nerve come laterally out of the spinal cord and pass through the foramen magnum, where they join to make the spinal accessory nerve.

Spinal Nerves

First, a quick refresher on spinal nerves. There's a diagram and explanation in one of my earlier posts on embryology. Thoracic spinal nerves (which have the white ramus going to the sympathetic trunk) are considered to be "typical" spinal nerves.

Now for a bit about the sympathetic trunk and referred pain! As I've mentioned before, the white ramus goes to the sympathetic trunk, which branches off into the grey ramus (to the body wall, where it supplies smooth muscle and glands in the skin) and visceral branches. Visceral branches lead to different locations depending on where they start. Sympathetic nerves arising from T1-T5 tend to ultimately innervate the head and neck, as well as thoracic organs. T5-T9 innervates the foregut (essentially all the digestive stuff before the intestines. Thanks, Google Images). T9-T11 innervates the midgut (small intestine, ascending colon, first 2/3 of the transverse colon) and gonads. Finally, T11, T12 and L1/2 innervate the hindgut (the rest of the large intestine) and the pelvic organs. These give rise to segments, or dermatomes, from which pain is "felt" due to sensory nerves from these locations returning with the sympathetic nerves. This is why heart pain is sometimes felt down the arm.

Analysing Gene Expression and its Regulation

Just realised that I missed out this transcription lecture. Joy. At least we got a laugh at how the slides refer to the gene that you're studying as "Your Favourite Gene," and even gave it the acronym YFG, as though it was a scientific term that everyone should know.

Describe how mRNA is isolated from total RNA.

This is reasonably simple, given that mRNA has a poly-A tail, which other types of RNA do not. Hence, mRNA can be isolated by using a cellulose matrix attached to oligo dT chains. This is done in a chromatography column with 0.5M NaCl.

Describe the steps in Northern blotting, including probe synthesis.

Northern blotting is a technique used to identify transcripts (i.e. mRNA molecules). I've written about it before in a previous post about hybridisation techniques.

As also mentioned in that previous post, probes use colour change or radioactivity in order to be identified. Molecules such as digoxigenin can be attached to the base (in areas where it will not interfere with the base pairing). Antibodies with fluorescent tags can then bind to these areas. In radiation, the alpha phosphate (i.e. the phosphate attached directly to the 5' carbon) is radioactive. (It has to be this particular phosphate, as the beta and gamma phosphates are lost as pyrophosphate in the DNA synthesis reaction.) Radioactive probes can then be identified via autoradiography.

Describe the type of quantification that can be obtained from Northern blots.

Quantification obtained from Northern blots is relative quantification. I presume this means that it isn't used to determine exactly how many mRNA molecules there are, but rather how many there are in comparison to other samples. Essentially, the denser the labelled bands on the nitrocellulose membrane, the more target mRNA there is. The density of the bands can then be compared to determine which samples have more or less of the target mRNA.

Describe the steps in in situ hybridisation, microarrays and RT-qPCR and the information obtained from each of these technologies

In situ hybridisation, as mentioned in my previous post about hybridisation techniques, determines where transcripts are found within tissues or cells. To prepare the slides, tissues are chemically preserved and embedded in wax, the tissue is sliced thinly and attached to slides, and the wax is removed. The other steps (probe generation, pre-hybridisation, hybridisation, washes and detection) are similar to that of Northern blots.

Microarrays are a method used to analyse the expression of thousands of genes simultaneously. They can also be used to compare the expression of genes in two different populations. Step number 1 is to isolate the mRNA, while step number 2 is to convert the mRNA into cDNA using reverse transcriptase. During this process, nucleotides are labelled with a fluorescent dye. Different dyes are used for the different populations. cDNA molecules are then hybridised to probes on the chip. (Different spots on the chip represent different genes- these are determined beforehand.) The colour and intensity of fluorescence at each spot on the chip gives relative quantification of gene expression in the two populations. Microarrays can also be used to perform cluster analysis, which is identifying genes that show similar expression patterns under similar conditions.

RT-qPCR, or reverse transcription quantitative PCR, continues on from our good friend PCR (see this earlier post for more details). This works pretty much like normal RT-PCR, except fluorescent tags are used to synthesise the new cDNA molecules. The intensity of the fluorescence increases as the number of cycles increases due to the production of more and more products containing fluorescent tags. This fluorescence can be quantified (presumably with some kind of machine that can detect the fluorescence) and a "threshold" intensity level is used for comparing different samples. RT-qPCR is also done with standards of a known concentration in order to create a standard curve. This standard curve can then be used to determine the original concentration of the mRNA of interest. (Let me know if that was confusing and I'll try and explain it better.)

Describe the function of pre-hybridisation and what factors affect stringency in hybridisation techniques such as Northern blotting, in situ hybridisation and microarrays.

The slides seem to take "pre-hybridisation" to mean "blocking" of the nitrocellulose membrane (or whatever medium you're using). "Blocking" basically involves adding proteins such as casein (found in milk) in order to cover the membrane and stop probes from binding to it (since it's better if the probe just binds to the mRNA of interest and not the membrane).

"Stringency" is a term that basically refers to how "strict" the probe is with regards to binding: will it bind only if there is an exact match, or is it a bit lenient? One factor that affects stringency is temperature: at higher temperatures (but below the melting temperature of the mRNA/probe complex), nothing will anneal except for exact matches. As you decrease the temperature, more and more probes will anneal. A second factor that affects stringency is salt concentration, as positive ions tends to stabilise the mRNA/probe complex (due to association with the negatively-charged phosphate backbone). The lower the salt concentration, the less likely the probe will bind unless it's a perfect match; the opposite is true for a high salt concentration.

So that's transcription over! w00t! Now for half a semester on proteins and enzyme kinetics... :P

Receptors and Other Drug Targets

Last post before the test! Joy :(

For each of the 4 superfamilies of receptor, be able to describe major characteristic features including
–  mechanism of signal transduction
–  receptor location
–  effector protein(s)
–  time scale of action
Provide at least one detailed example of a drug that acts via each of the 4 receptor superfamilies 

Ion-channel receptors

Ion-channel receptors, as their name suggests, are receptors that are ion channels. When activated by an agonist (usually a fast neurotransmitter), they open, allowing ions to flow through them. (Ions generally do not cross the cell membrane as they are charged particles.) They are located in the cell membrane, with both their C and N-termini located extracellularly. As ion flow occurs rapidly, their mechanism of action likewise occurs rapidly.

One example of a drug that acts on ion-channel receptors is Pancuronium. It antagonises nicotinic ACh receptors, which are also Na+ channels. Since it is an antagonist, it prevents the Na+ channels from opening and allowing Na+ from crossing the cell membrane. This stops neurons from producing their action potentials, and thus results in local anaesthesia.

G-protein coupled receptors

G-protein coupled receptors have their effects by interacting with G-proteins, which in turn react with other second messengers in the cell. I've spoken about G-proteins before, but just a quick recap: G-proteins are proteins that bind GTP (guanosine triphosphate). When this GTP is hydrolysed, only GDP (guanosine diphosphate) remains. G-protein coupled receptors act as GEFs (guanosine exchange factors), which, when bound by an agonist, get rid of the GDP on the G-protein so that it can be replaced with a fresh GTP molecule, thus activating the G-protein. G-proteins have three subunits (alpha, beta and gamma) which can interact with other effector proteins that release second messengers (for example Gα_s activates adenylate cyclase, which produces cAMP).

G-protein coupled receptors are also located in the cell membrane. However, while their N-terminus is also located extracellularly, their C-terminus is located intracellularly. The intracellular region reacts with the G-protein.

Most G-protein coupled receptors respond to hormones or slow neurotransmitters. As more steps have to take place for them to have their effect (they have to activate the G-protein, which in turn has to react with other stuff), their method of action is relatively slow compared to ion-channel receptors. However, in the whole scheme of things, they are considered to be fast-acting (within seconds).

Now for an example! Salbutamol is a beta 2-adrenoceptor agonist that relieves bronchospasm in asthma. Beta 2-adrenoceptors are G-coupled receptors that, when activated, release a stimulatory G protein (G_s) which activates adenylate cyclase. cAMP is then produced, resulting in relaxation of the airways.

Enzyme-linked receptors

Enzyme-linked receptors pretty much are enzymes. Like G-protein coupled receptors, they are located in the cell membrane, with their N-terminus extracellular and their C-terminus intracellular. Usually the N-terminus is where the signalling molecule binds, whereas the C-terminus is the enzyme part. They usually respond to hormones for growth and differentiation, and as such their effects are slower, normally in the time scale of minutes.

An example of a molecule that binds to an enzyme-linked receptor is insulin. Insulin binds to insulin receptors, which act as tyrosine kinases. The overall effect of these kinases is to translocate the glucose transporter GLUT4 to the cell membrane, so that glucose can enter the cell where it is needed for metabolism.

DNA-linked receptors

DNA-linked receptors act directly on DNA (again, as their name suggests... methinks these receptors won't be difficult to remember). They are the only receptors that are located intracellularly. DNA-linked receptors normally respond to hormones, particularly steroids. Their mechanism of action can take hours as genes have to be transcribed and proteins have to be produced.

An example here is glucocorticoid drugs such as cortisone. They are anti-inflammatory agents that have their effects by binding to DNA-linked receptors, which in turn bind to the DNA. (As for which genes they transcribe... I guess I'll have to find that out.)

Explain, with examples, how ion channels, enzymes and transporters are important drug targets.

I feel like I've already done this by talking about the receptors above, but they did provide some more examples in the lecture so let's go over those.

Ion channels

Some of the drugs that react with ion channels include blockers and modulators. Blockers physically plug the channel, preventing stuff from passing through. I've already given an example here with local anaesthetics blocking nicotinic ACh receptors (which are also Na⁺ channels). Modulators bind to other accessory sites on the channels, modulating their activity. An example here is benzodiazepines (e.g. Valium) which are sometimes prescribed for anxiety. They enhance the opening of GABA-activated Cl^- channels.

Enzymes

Once again, I feel like I've pretty much covered this in the enzyme-linked receptor section, so I'm just going to provide a few more interesting tidbits instead. Interesting tidbit number 1 involves substrate analogues, which are basically drugs that act as competitive inhibitors. Sometimes the enzyme might actually break them down, but an abnormal metabolite is produced. For example, fluorouracil, an anti-cancer drug, replaces uracil. It cannot be broken down to thymidylate (a component of DNA), so DNA synthesis is inhibited.

Transporters

Sometimes transporters are also targets for drugs. For example, SSRIs (selective serotonin reuptake inhibitors) selectively prevent serotonin from being transported back into a neuron, allowing it to hang around in the synapse for longer and continue to stimulate the postsynaptic neuron. False substrates can be an issue here as well- amphetamines can hijack the noradrenaline transporter and replace or release noradrenaline and serotonin.

Transcription Regulation of Eukaryotic Gene Expression II

My last post was called Transcription Regulation of Eukaryotic Gene Expression I... well, this one is imaginatively called Transcription Regulation of Eukaryotic Gene Expression II!

Describe coordinated and combinatorial control of eukaryotic gene expression, and give examples.

I've mentioned combinatorial control of eukaryotic gene expression in my previous post. Essentially, the presence of different combinations of gene regulatory proteins may affect transcription. Which proteins are present or absent may depend on the cell and the stage of development. As well as multiple proteins controlling the expression of a gene, individual proteins may also contribute to the expression of several different genes. For example, the glucocorticoid receptor (which is actually a transcription factor) can bind to several different genes and play roles in their activation.

Describe the functions and structures of RNA polymerase I, II and III.

Yup, I'm finally going to talk about RNA polymerases I and III. Time to give them some love. (RNA Polymerase II is still the star of the show though. Just because. Okay, well, their roles in transcribing mRNAs which end up as proteins is pretty damn important, I suppose.)

First, a word on their functions. RNA polymerase I is mainly responsible for transcribing most pre r-RNA ("pre" refers to RNA that hasn't undergone post-transcriptional processing yet). RNA polymerase II, as I just alluded to, transcribes mRNAs, as well as snRNAs (small nuclear RNAs) and miRNAs (micro RNAs). (According to one of my previous posts, snRNAs process RNA transcripts while miRNAs cause degradation or block translation of RNA. Finally, RNA Polymerase III transcribes tRNAs and other small RNAs that aren't transcribed by the other two.

Now for the structures! All three RNA polymerases have 5 subunits, just like how prokaryotic RNA polymerase has 5 subunits. RNA Polymerase II's alpha-like subunits are quite different to those of the other two, however. It also has a C-terminal domain (CTD) on one of its beta-like subunits. I've written about the C-terminal domain in an earlier post. Essentially, it contains almost perfect repeats of seven highly conserved amino acids, some of which can be phosphorylated, allowing capping proteins and so forth to "dock" and do their job.

Describe the factors and assembly of RNA polymerase I and III transcription initiation complexes.

RNA Polymerase I, like II, is attracted to particular regulatory sequences. These consist of a core element and an upstream element which stimulates transcription. I'm not sure how much detail we need to know about this, however, as there is plenty of detail on the slide but I don't think it was covered in such great detail in the lecture. The gist of it though is that upstream activating factor (UAF), core factor (CF) and so forth assemble on the upstream element and/or the core element, positioning RNA polymerase I and allowing it to do its job.

RNA polymerase III's promoter regions are a bit different in that they are located inside the sequences that are to be transcribed. These regions are known as A, B and C boxes. A and B boxes are located in all tRNA genes, whereas the C box is located in the 5S-rRNA gene. (5S-rRNA is one of those rRNAs that is made by pol III and not pol I. I'm just going to call them "pols" from now on because I'm sick of typing out "polymerase.") Just like the other two RNA pols, pol III has transcription factors, such as TFIIIA, TFIIIB and TFIIIC. (Wow, how imaginative.) TFIIIA is only found in the 5S-rRNA gene, whereas TFIIIB and TFIIIC are found in all tRNA and 5S-rRNA genes. TFIIIB is the one that has the TATA-binding protein (TBP)- yes, apparently pol III needs a TBP as well.

Describe the structure and function of mitochondrial and chloroplastic RNA polymerases, and their transcription initiation complexes.

As you should know by now, mitochondria and chloroplasts were probably originally bacteria that moved into their eukaryotic hosts. As such, they have circular genomes, like bacteria. They also have their own polymerases.

Human (and probably other eukaryotic) mitochondrial DNA (mtDNA) has two strands: a heavy strand and a light strand. Most genes are located on the heavy strand. The two strands are transcribed in opposite directions by mitochondrial RNA polymerase, a.k.a. POLRMT. POLRMT is a bacteriophage-type RNA polymerase (whatever that means). Main point to know about this one is that POLRMT is actually made outside of the mitochondria: its gene is transcribed in the nucleus and translated in the cytoplasm, just like most other proteins. It is then imported into the mitochondria.

POLRMT is placed at the promoters by the transcription initiation factors TFAM and TFB2M. (Just looked up what they stand for- TFB2M stands for Transcription Factor B2, Mitochondrial, so I assume TFAM stands for Transcription Factor A, Mitochondrial.) The promoters also have names: the light strand promoter is LSP, whereas the heavy strand promoter is HSP1. (Not sure if there's an HSP2.)

Chloroplasts, like mitochondria, have circular genomes with strands transcribed in opposite directions. However, transcription occurs in blocks, each with a different promoter, as opposed to mtDNA where the entire strand is just translated in one go. Chloroplasts have two types of RNA polymerases: NEP (nuclear-encoded polymerase) which is encoded in the nucleus, and PEP (plasmid/chloroplast-encoded polymerase), which is encoded in the chloroplast. NEP is required for transcription of 3 of the 4 core subunits of PEP. NEP and PEP transcribe different genes.

Transcriptional Regulation of Eukaryotic Gene Expression I

Yup, still rambling...

Describe gene control regions found in eukaryotes, e.g. promoters, proximal promoter elements, enhancer elements

Describe eukaryotic general transcription factors, mediators, chromatin remodelling complexes

Promoters

Promoters are basically sequences on the DNA that tell the transcription factors and polymerase where to bind. One common promoter sequence is the TATA box, so called because it has lots of T and A residues. One of the main proteins that binds here is TBP (TATA-binding protein), which is a subunit of a larger transcription factor called TFIID (the TF stands for "transcription factor," the II refers to RNA Polymerase II and the D stands for "distortion" as it "distorts" the helix). Aside from TFIID, there are other transcription factors that bind here, such as TFIIH which has some helicase activity (hence the H for "helicase"). Most of these general transcription factors (GTFs) dissociate when elongation begins.

Proximal promoter elements

Proximal promoter elements are regions near the promoter that also help to stimulate transcription. They do this via the proteins that bind to them.

Enhancer elements

Enhancers are kind of like proximal promoters in that they help to stimulate transcription, but they do their job from a distance. They can be thousands of base pairs away, whereas proximal promoter elements are only 100-200 base pairs away.

Mediators

Mediators are large complexes with roughly 30 subunits. They help to mediate transcription by "bringing together" all of the transcription factors at the promoter, proximal promoter elements and enhancer elements. They also bind RNA Polymerase II. (I'll cover the other RNA Polymerases in a later post.)

Chromatin remodelling complexes

As you should know by now, DNA doesn't just exist as a big long double helix in the nucleus- it has to be packaged well in order to fit. DNA is wound around other proteins such as histones. The complex of DNA and protein is known as chromatin. How tightly or loosely the chromatin is packaged can affect transcription: it is harder for other proteins to bind to DNA when it is tightly packaged. Chromatin remodelling complexes can influence transcription by influencing the packaging of the DNA.

Describe how some eukaryotic gene activator proteins change chromatin structure

I've just mentioned chromatin remodelling complexes, which change interactions between the DNA and histones. (Histones are basically sets of 8 proteins- an octamer- bound together. DNA can wrap around them, like thread around a spool.) As I said before, they can make chromatin more tightly packaged (heterochromatin) or more loosely packaged (euchromatin). The former decreases transcription, whereas the latter process increases transcription.

Aside from chromatin remodelling complexes, there are other proteins that can affect chromatin structure. One of these is histone chaperones, which can add or remove histones. Histone-modifying enzymes can also change groups on the histone proteins (i.e. modify the side chains of their amino acids). All of these can make the DNA more or less accessible to the transcription machinery (i.e. all of the proteins that regulate transcription). Alternatively, the acetyl or methyl groups may provide markers that transcription factors can bind to.

Describe eukaryotic gene repressor protein operation

There are many ways in which gene repressor proteins can work:

1. Competitive DNA binding. In this case, the repressor protein binds to the same spot that an activator would bind. This prevents the activator from binding and hence prevents transcription.
2. Masking the activation surface. In this case, the repressor protein binds to the activator protein, stopping it from binding to the mediator protein (or whatever other protein it may need to bind to in order to activate transcription).
3. Direct interaction with the general transcription factors. This is kind of similar to #2, but with other proteins such as mediators. This action can also prevent activator proteins from binding to the mediators.
4. Recruitment of chromatin remodelling complexes. Essentially the repressor just harnesses the action of the chromatin remodelling complex to package the DNA so that it cannot be accessed by transcription factors.
5. Recruitment of histone deacetylases. Deacetylation of histones generally leads to reduced transcription.
6. Recruitment of histone methyl transferase. The methylation state of histones can also affect transcription.

Describe cooperative binding of eukaryotic gene regulatory proteins and how this affects gene expression

Cooperative binding of eukaryotic gene regulatory proteins is somewhat like combinatorial control in prokaryotes- see my previous post. Sometimes particular combinations of proteins may attract coactivators or corepressors, each of which obviously has a different outcome on transcription.

Describe techniques/technologies used to identify gene regulatory regions and proteins

There are several different techniques and technologies that can be used to figure out the location of potential gene regulatory regions.

The first one, deletion analysis, is sometimes known more colloquially as "protein bashing" as it is a bit cruder than other methods. The first step in this method is to isolate an area upstream of the gene, as this is the area that usually contains regulatory sequences. The next step is to use restriction enzymes to cut it to give you strands of different lengths- known here as a 5'-deletion series (as it is the area upstream of the 5' end). These can then be ligated into plasmid vectors in E. coli in order to amplify and isolate the plasmids. These plasmids also contain a "reporter gene" with products that can be measured. After cultured cells have been transfected with plasmids, gene expression can be measured. Regulatory regions can be identified from there.

The next method is called DNA footprinting. Again, this involves isolating the upstream region of a gene and cleaving it with enzymes. Before cleavage, however, proteins are allowed to bind. Digestion will not occur where proteins are bound. Proteins are then removed and fragments are run through a gel (I've mentioned electrophoresis on an earlier post) and, if regulatory regions are present, there should be some areas where no cleavage is observed (as noted by some short fragments, then suddenly some longer fragments- with nothing in between). This is known as a DNA "footprint." The region can then be isolated, and the sequence can be determined.

The third and final method that I'm going to talk about is Electrophoretic Mobility Shift Assay, which thankfully has an acronym- EMSA. It's somewhat similar to DNA footprinting, except that the proteins are not removed before running through the gel. (DNA without proteins attached is also run through the gel as a control.) DNA attached to proteins does not travel as far as DNA that isn't attached to anything. Also, the larger the attached proteins, the slower the DNA will run through the gel. This method therefore gives indications of where proteins are and how large they are.

After EMSA is complete, the regulatory proteins can be identified through another process. The cell extract is fractionated in column chromatography, where different proteins pass through the column at different rates and can therefore be eluted separately. There's a couple more steps after this- namely, running aliquots of the eluted fractions through gel again and then going back to the fractions that produced a shift to purify the proteins- but I'm still not quite clear on why these are necessary. I'll have to get back to you another time on this one.

Transcription Factors

Describe the general characteristics of gene regulatory proteins, including binding sites and interactions made with target DNA

By "gene regulatory protein," I'm going to assume that we mainly have to learn about transcription factors, since transcription is the stage at which most gene regulation takes place.

Transcription factors have two domains: a DNA-binding domain, and an activation or repression domain. The former usually binds to the major groove of DNA, whereas the latter usually binds to other proteins to regulate transcription. Be aware that the bonds to DNA are not via base pairs (since proteins don't have bases that can pair with the DNA)- instead, they bond through hydrogen bonds, ionic bonds, hydrophobic interactions and so forth.

Describe the specific structural components, DNA interactions, and dimerisation ability of helix-turn-helix, zinc finger, leucine zipper, helix-loop-helix, two-stranded beta-sheet, loop region

These few paragraphs are to do with different "motifs," or types of structures, that you might see in regulatory proteins.

Helix-turn-helix: these are, as the name suggests, two alpha helices with a short chain (the "turn") between them. The helices are held at a fixed angle due to the interactions between them. One of the helices, usually the more C-terminal one, is the "recognition helix" that fits into the major groove of the DNA. The other helix (the more N-terminal one) tends to have more structural and positioning roles. Often two transcription factors with the helix-turn-helix motif will bind to the DNA as a dimer. As there are more contacts with the DNA due to the dimerisation, there is an increased binding affinity with the DNA.

Zinc finger: these are "finger-shaped" structures that include at least one Zn2+ ion. They are defined by the residues that associate and coordinate the Zn2+ ion. Common groups are Cys-Cys-His-His (C2H2) or Cys-Cys-Cys-Cys (C4) zinc finger motifs. The Zn2+ ions help to stabilise the "finger" structure. One example of a transcription factor containing the Zn2+ motif is the glucocorticoid receptor (so-called because they thought it was a boring old receptor before they figured out that it was a transcription factor). It contains a C4 zinc finger motif. It can bind glucocorticoid steroid hormones such as cortisol, which are produced during starvation and high physical activity. When bound, it can act as an activator, stimulating the transcription of genes that increase glucose production in the liver.

Leucine zipper: these consist of two alpha-helices that are coiled around each other (the "coiled-coil" structure). Each alpha helix has a hydrophobic leucine residue at every seventh position, allowing for hydrophobic interactions between the helices. The two helices work together as a dimer that contacts two adjacent major grooves.

Helix-loop-helix (HLH): these are somewhat similar to helix-turn-helix in that they consist of two alpha helices, but there are some differences. Firstly, while helix-turn-helix proteins tend to bind to the DNA at their C-terminal helix, the C-terminal helices of helix-loop-helix motifs instead form a coiled-coil structure (HLH motifs can form homodimers or heterodimers). It's the N-terminal helices that contain basic amino acids that interact with the DNA. I'll probably have to find out a bit more about the differences between HLH and helix-turn-helix motifs, as I'm pretty confused at the moment.

Two-stranded beta-sheet: as the name suggests, their binding sites are beta sheets rather than alpha helices. Like other motifs, two-stranded beta-sheets often work in dimers, with each monomer contributing one strand to interact with the major groove of the DNA.

Loop regions: these are simply regions that don't have a defined structure like alpha helices or beta sheets. Some of these regions are also capable of recognising major and/or minor grooves.

Describe combinatorial control involving gene regulatory proteins

Combinatorial control involves the bringing together of different transcription factors. Several different monomers may recognise the same DNA sequence, but the combinations in which they bind may affect transcription. Alternatively, different monomers may recognise the different DNA sequences in a given area. The monomers themselves may be expressed only in particular cells at particular times, allowing for combinatorial control of transcription.

Regulation of Gene Expression in Prokaryotes

LMS is currently down (due to a lightning strike or something), so at first I thought that it'd be a good excuse to not study. But then someone pointed out to me that the app (Blackboard Mobile Learn) is still useable, so here I am.

Describe and give examples of positive and negative regulation of prokaryotic gene expression

I've done this before in two previous posts: The Lactose Operon and Positive Regulation of the Lactose Operon.

Describe and give examples of how some bacterial gene regulatory proteins can act as repressors or activators

Well, since nothing in the body likes to be simple, proteins can have different functions depending on where they bind and so forth. If a protein binds in a way that makes it harder for the polymerase to get to the gene, then the protein is acting as a repressor. If the protein binds in a way that facilitates the polymerase binding to the gene, then the protein is acting as an activator.

The bacteriophage lambda repressor is an example of a regulatory protein that can act as either a repressor or an activator (which is pretty confusing, given the name). Like other proteins, it can repress by preventing binding of RNA polymerase, or activate by facilitating binding of RNA polymerase.

Describe and give examples of the roles of alternative sigma factors in prokaryotic gene expression

I've mentioned sigma factors in an earlier post, but just a quick refresher: in prokaryotic cells such as E. coli, the core enzyme of RNA polymerase has five subunits, which a special protein known as sigma factor can bind to in order to form a holoenzyme (core enzyme plus sigma factor). Ultimately, it's sigma factor that helps the polymerase to bind to the promoter. The sigma factor dissociates soon after transcription has begun.

There are different sigma factors which recognise different promoters. This helps to coordinate transcription of different genes. The most common sigma factor is sigma 70. Another important sigma factor is sigma 54, which tends to bind to genes that have enhancer regions (i.e. activators far away from the transcription start site).

Describe and give examples of two-component regulatory systems in prokaryotes

A "two-component regulatory system" is, to my understanding, essentially the use of two components (a sensor and a response regulator) to regulate genes. The sensor protein senses a change in the environment, which causes it to react with the response regulator protein (usually by phosphorylation, but there are other ways), which in turn causes a gene to be activated or repressed. The functional domain of the sensor protein is called the transmitter domain, and it's responsible for phosphorylating or otherwise activating the response regulator. The functional domain of the response regulator protein is called the receiver domain, and it's the part that gets phosphorylated or whatever.

Example number 1: NtrB and NtrC. NtrB is a kinase (i.e. a protein that tacks phosphate groups onto other stuff) that is activated when glutamine levels are low. When glutamine levels are low, NtrB phosphorylates NtrC, which stimulates the transcription of the gln gene which codes for glutamine synthetase. Glutamine synthetase, as its name suggests, catalyses the synthesis of glutamine, restoring glutamine levels in the cell.

Example number 2: PhoR and PhoB. PhoR is a transmembrane protein which senses phosphate in the periplasmic space (i.e. between the inner and outer membranes). When phosphate levels in the periplasmic space is high, some of it binds to the periplasmic domain of PhoR, keeping it inactivated. When phosphate levels drop, however, phosphate dissociates from PhoR, activating it. PhoR then transfers a phosphate from ATP to PhoB, a protein in the cytosol. This activates PhoB, allowing it to act as a transcription factor for several genes.

Describe and give examples of phase variation as a mechanism controlling gene expression

Phase variation basically involves inversion of genes (i.e. taking it out and putting it back in backwards). I've mentioned inversion briefly in an earlier post. The consequences of inversion include "switching off" promoters, if it's a promoter region that is inverted. Phase variation isn't a very common way of controlling genes, but it does happen.

An example in where phase variation is used is in flagellin genes in salmonella. Salmonella has plenty of flagella that help it to move around, but these flagella make them way too obvious to immune system cells in people that they're trying to kill. Phase variation, which switches off the H2 flagellin gene, allows them to sneak around much more effectively.

Introduction to Drug Action

Yet another pharmacology post! I've decided that I really don't get the order in which these lectures have been in... but oh well.

Be able to answer the question “What is a receptor?”

A receptor is basically something that a drug or another signalling molecule can bind to. When it is bound (or, in some cases, when it isn't bound), it induces some kind of effect. See my earlier post on Biochemical Messengers for more information.

Be able to explain concepts such as agonism, partial agonism and antagonism in terms of drug affinity and efficacy

Firstly I'll explain what "affinity" and "efficacy" mean. "Affinity" refers to a drug's ability to bind to a target. "Efficacy" refers to the degree of receptor activation- a high efficacy means a high activation of receptors.

An "agonist" is a drug that binds to a receptor to induce an effect. Agonists have affinity and efficacy. An antagonist is the opposite: it also binds to a receptor, but it does not cause an effect: instead, it blocks other agonists from having a potential effect. (Rude.) They have affinity, as they do bind to the receptors, but they have no efficacy. A partial agonist is somewhere in between: it causes some effect, but not as much as a full agonist. They also have affinity, but low efficacy.

Be able to explain what is meant by drug selectivity

Drug selectivity, as the term suggests, refers to a drug binding to certain targets but not to others. This is often to do with the structure of the drug. See my previous post on structure-activity relationships for more details.

Be able to describe the relationship between agonist concentration and receptor occupancy or response

Generally, as the concentration of an agonist increases, the number of receptors that are occupied by said agonist also increases. Because of this, the response also increases. The concentration at which half the maximal response is achieved is known as EC50. If a drug has a low EC50, it is said to be more potent; if a drug has a high EC50, it is not as potent. Potency depends largely on affinity and efficacy.

Be able to explain the differences between reversible and irreversible receptor antagonism

Antagonists come in two main types: those that bind temporarily and dissociate (known as "reversible antagonists") and those that get stuck there forever (also known as "irreversible antagonists").

When a reversible antagonist binds, the concentration-effect curve is "shifted" to the right. This means that EC50 is higher, which in turn means that more agonist is required in order to "compete successfully" for receptors. The size of this "curve shift" depends on several factors such as the affinity and concentration of the antagonist.

When an irreversible antagonist binds, there are fewer spaces in which an agonist can occupy. The overall effect is just like having fewer receptors: the maximum effect is decreased, and the slope of the curve is also reduced. Once again, this "curve shift" depends on factors such as concentration.

Stereochemistry

This *should* be a real quickie, given that it draws on concepts I've talked about before (namely chirality and structure-activity relationships). (Strangely, I don't have a post entirely on chirality- probably a good post would involve diagrams which I'm too lazy to draw- but that post I just linked to, as well as this post on carbohydrates, cover a fair bit.)

1) Define the terms “stereoisomer” & “chiral centre"

Ehhhh can't be bothered typing, read this post on carbohydrates instead.

2) Demonstrate a basic appreciation of the drawing conventions used to denote the presence of a chiral carbon within drug structures

In diagrams, you might have seen dashed lines and/or wedges between atoms. Dashed lines indicate that the atom in question is going "into" the page (i.e. away from you) while wedges indicate the opposite- that the atom in question is coming "out of" the page. You can use these to help you work out how the atoms are oriented, and from that you can work out which enantiomer they are. In racemic mixtures (i.e. mixtures with both enantiomers in equal quantities), a squiggly line might be drawn instead.

3) Show an appreciation of the pharmacodynamic implications of stereoisomerism, using the “3 contact point model” to explain such phenomena

Stereoisomers have different shapes, which means that they have different affinities for different targets. Can't be bothered drawing models or anything, unless anyone really wants me to.

4) Show an appreciation of the pharmacokinetic implications of stereoisomerism in drugs, especially during drug metabolism.

Once again, different stereoisomers have different affinities for different targets. Not only does this affect how well a drug binds to its target, but it may also affect absorption because certain stereoisomers may have greater affinities for certain transport proteins. (Note that chirality only affects "active" processes such as active transport- it doesn't affect a drug's ability to diffuse across the membrane.)

Another important point of note is that sometimes metabolism affects stereoisomerism. Metabolism can change a drug from one stereoisomer to another, abolish chirality or establish new sites of chirality. These all have implications for how a drug acts in the body.

5) Be able to define the term “chiral switching” and give examples of the use of this strategy.

"Chiral switching" is basically a company marketing a pure enantiomer of a drug, rather than the racemic mixture. For example, citalopram, an antidepressant drug, is a racemic mixture; escitalopram contains the S-enantiomer of citalopram only (hence escitalopram. Very funny, pharmacists). This is often done if one enantiomer is known to be much more effective than the other. Selling a drug containing only the effective stereoisomer means that only half the dose needs to be taken, while eliminating any negative effects that metabolism of the ineffective stereoisomer may have had. Going back to the escitalopram example, apparently escitalopram may be more effective than citalopram. (On an unrelated note, escitalopram is a massive pain in the rear end to go off. I'm currently tempted to stick a meme on here with my psychiatrist's face and the words "'Go off your escitalopram,' she said. 'It will be easy,' she said." But I'm nice, and I won't do that to her.)

A few quick definitions: if one enantiomer is better, then the better one is known as the eutomer while the worse one is the distomer (from the Greek "eu" meaning "good" and "dis" meaning "bad"). The ratio between the two is known as the eudismic ratio.

6) Show an appreciation of the toxicological implications of stereoisomerism

Of course, if stereoisomerism can have implications as to which targets drugs bind to, it can also have toxicological implications if the binding of one stereoisomer leads to negative effects. The no-brainer solution then is to use "chiral switching" to give patients only the stereoisomer that isn't toxic; however, this isn't always so simple as chirality may change within the body due to metabolism or otherwise.

One possible example of so-called "chiral toxicity" is ketamine. It's an anaesthetic, but it's also used as a recreational drug. It's thought that S-Ketamine has better anaesthetic activity and fewer side effects, while R-Ketamine is more likely to cause psychosis, agitation and amnesia.