Cardiac Output and Blood Pressure

Second lecture of seven! (Wow, can't believe we did the heart for so long... 0_o)

List the compartments of the cardiovascular system.

Heart, blood vessels, blood. Have I missed anything?

Define Heart Rate, Stroke Volume, Cardiac Output and Venous Return.
Know typical values for Heart Rate, Stroke Volume and Cardiac Output.

• Heart Rate: The number of times the heart pumps every minute. Usually around 70bpm at rest.
• Stroke Volume: The volume of blood pumped per beat. Usually measured in millilitres, and is usually around 70mL.
• Cardiac Output: The amount of blood pumped around the body in one minute. This is equal to the heart rate multiplied by the stroke volume, and is usually around 5L/min.
• Venous Return: This is the same as cardiac output, because you don't want more or less blood coming in as compared to what leaves the heart (or you'd have blood accumulating somewhere). Hence, this is usually around 5L/min as well.

List the variables which alter cardiac output and make calculations from CO = HR x SV.

As I just mentioned, the cardiac output is simply the heart rate multiplied by the stroke volume. Hence, anything that affects the heart rate and stroke volume (stimulation by the autonomic nervous system etc.) will likewise affect cardiac output. Venous return is one way in which cardiac output can be affected, as we shall see...

Explain the interaction among venous return and cardiac output for the left and right heart.
Describe the Frank-Starling law of the heart and sketch the cardiac function curve.

According to the Frank-Starling law of the heart, "within physiological limits the heart pumps out all of the blood that it receives." Hence, the more blood that returns to the blood via the veins, the larger the stroke volume. This is because the extra blood stretches the ventricles, allowing them to generate more force. This is kinda like an elastic band: if you stretch an elastic band a little bit, it won't really snap back. If you stretch an elastic band a lot though, it'll snap back hard.

This relationship can be displayed in the Cardiac Function Curve (which is probably the blandest graph I've ever drawn, and that's saying something):

Define Systolic, Diastolic, Mean and Pulse Pressure for the systemic arterial system.
Know typical values for systemic arterial systolic, diastolic, mean and pulse pressure.
Calculate mean and pulse pressure from systolic and diastolic pressure.

• Systolic Pressure: The highest systemic arterial pressure, attained when the heart contracts. This is usually around 120 mmHg.
• Diastolic Pressure: The lowest systemic arterial pressure, attained when the heart relaxes. This is usually around 80mmHg.
• Mean Pressure: The average pressure across the cardiac cycle. This is usually around 90-95mmHg. This is because the heart spends 2/3 of its time in diastole and only 1/3 of its time in systole, so to measure mean arterial pressure you'll need to take 1/3 of the systolic pressure and 2/3 of the diastolic pressure and add these values together.
• Pulse Pressure: The difference between systolic and diastolic pressures. Usually around 40mmHg.

Describe the systemic and pulmonary circulations and explain the difference between them.

The systemic circulation pumps blood from the left side of the heart to the whole body (except for the lungs). The blood in the systemic circulation is oxygenated when it leaves the heart, and loses oxygen to the tissues as it travels. Blood is deoxygenated by the time it comes back to the heart. This system is relatively high pressure, with a pressure of around 120/80 mmHg, as mentioned earlier.

The pulmonary circulation pumps blood from the right side of the heart to the lungs. This blood starts off deoxygenated, but becomes oxygenated as it passes through the lungs. This system is relatively low pressure (shorter vessels = less resistance due to less overall SA... I think), with a pulmonary artery blood pressure of around 25/8 mmHg.

Composition of the Blood

Onto another topic!

Define TBW, ECF, ICF, IF, Lymph, Plasma and Blood
Calculate TBW, ECF, IF and blood volume from sex and body weight
Define and know typical values for PCV 

• TBW: Total Body Water. It's... the total of all of the water in your body, I guess. This is usually around 60% of your weight for males and 50% for females (females have more fat so we get nicer curves :) ). Expanding on that a bit, overweight people will have a lower TBW for their weight, and vice versa.
• ECF: Extracellular Fluid. This is all of the fluid that is outside of the cells. It is roughly 1/3 of your TBW.
• ICF: Intracellular Fluid. This is all of the fluid that is inside the cells. It is roughly 2/3 of your TBW.
• IF: Interstitial Fluid. This is a subset of ECF- it's the ECF in solid organs. This is roughly 1/5 of your ECW, or 1/15 of your TBW.
• Lymph: Lymph is basically your ECF plus a few extra proteins. Essentially, extra water from your tissues is drained through the lymph system back into the venous system in order to be recirculated throughout the body.
• Plasma: Plasma is pretty much everything in blood except for the cells. It is straw-coloured and includes water, proteins, salts, nutrients, wastes and gases. Roughly 20% of ECF is plasma.
• Serum: Plasma without clotting factors.
• Blood: The red stuff that comes out when you get wounded. Need I say more?
• PCV (Packed Cell Volume): The percentage of blood volume that is made up of cells.

Know typical values for blood volume and PCV

I could've done this in conjunction with the list above, but I didn't want the list to be even more cluttered. Here's a separate list. Note that these are only a rough guide: these values vary from person to person, depending on gender, weight, fat content etc.

• TBW: roughly 42L
• ECF: roughly 14L
• ICF: roughly 28L
• IF: roughly 3L
• Blood volume: 5L
• PCV: 42% (female), 45% (male)

List the cells present in blood

More lists!

• Erythrocytes: red blood cells
• Leukocytes: white blood cells. Can be subdivided further into monocytes, eosinophils, basophils, neutrophils or lymphocytes, as I'll explain later.
• Platelets

Describe the role of erythrocytes in the transport of O₂ and CO₂
List the components of haemoglobin and where O₂ binds

As I'm sure you know, erythrocytes, or red blood cells, are biconcave discs that look red (hence the name). They do not have a nucleus or organelles, but they have shitloads of haemoglobin, which is good, because haemoglobin is useful for carrying around O₂ and, to a lesser extent, CO₂.

So what is haemoglobin exactly? It's a protein, just like many other important things in the body. It is made up of four polypeptide chains (a.k.a. "globins"): two α and two β. Each chain is associated with a haem unit (hence the haem unit plus polypeptide chain = haemoglobin!). The main feature of the haem unit is that it contains Fe²⁺, which oxygen can reversibly bind to in order to be transported around the blood. This is how most oxygen travels around the blood- only a small amount (~1.5%) is actually dissolved in the blood.

Carbon dioxide is transported around the blood in several different ways. It is more soluble than oxygen, so around 5% of CO₂ is transported this way. Carbonic anhydrase in the red blood cells can also catalyse the reaction between CO₂ and water to form H⁺ and HCO₃^-, both of which can travel around in solution. A third way that CO₂ can travel around is by binding to haemoglobin or to other proteins in the blood in order to form carbamino compounds (e.g. carbaminohaemoglobin). It is important to note that CO₂ does not bind to Fe²⁺, but to amino groups in haemoglobin.

Now for a note on red blood cell production! Erythrocyte production is regulated by erythropoietin, or Epo for short. It's usually released from the kidneys in response to hypoxia (low oxygen conditions), but can also be stimulated by growth hormone, thyroid hormone or testosterone (I guess you need more blood as you grow and develop...).

List the leukocytes present in blood
Describe the main function of each class of leukocyte

I've already listed the leukocytes present in blood: monocytes, eosinophils, basophils, neutrophils and lymphocytes. Now it's time to talk about them in more detail!

Eosinophils, basophils and neutrophils are collectively known as granulocytes as they contain granules, which contain important chemicals. They are also known as polymorphonuclear leukocytes (poly = many, morpho = shape, nuclear = nucleus, so these leukocytes' nuclei have all kinds of weird and wonderful shapes), but that's a bit of a mouthful to say. Anyway, the three granulocytes are named after the dyes that stain them: eosinophils are stained by the dye eosin, basophils are stained by basic dyes, and neutrophils don't get stained by either.

That's all well and good, you might say, but what about the function of these cells? Neutrophils, the most abundant leukocytes, are normally the first to arrive at the site of an infection, and they are phagocytic (they eat cells), especially towards bacterial cells. Eosinophils are also phagocytic (though only weakly) and are able to destroy parasites, and also play roles in allergies and inflammation. Basophils also play roles in allergies and inflammation. (I assume we'd go into much more detail when we study immunology next semester.)

Now for the non-granular cells, or agranulocytes! These are the monocytes and the lymphocytes. Monocytes are cells that can become macrophages, which basically go around eating everything up. They can also release messenger molecules called cytokines, which enhance the immune response. Lymphocytes come in two main varieties (yup, subcategories of subcategories): B-lymphocytes and T-lymphocytes. B-lymphocytes (which make up roughly 20% of all lymphocytes) are the ones that produce antibodies- this is known as humoral immunity. T-lymphocytes, which are responsible for cell-mediated immunity, come in two varieties (yup, even more subcategorisation): cytotoxic T-cells (kill stuff) and helper T-cells (release cytokines).

Phew!

Describe the function and origin of platelets

Platelets are fragments of megakaryocytes, which are large cells found in the bone marrow. As they are only fragments of the cytoplasm of megakaryocytes, they contain some organelles, but have no nuclei. They adhere to damaged blood vessel walls, and clump together to plug up the gap. They also release growth factors that help repair, as well as help coagulation enzymes form insoluble strands of fibrin to hold the clot together.

Wow, that was long... and we haven't even gotten into the nitty gritty of how everything works yet!

Control of Ventilation

Time to give physiology some love! I haven't really posted much about physiology throughout the semester, but since it's my first exam, I guess it's time to get right back to it!

Since I was blogging about respiratory physiology before, I'm going to finish off that topic with this post on control of ventilation.

List the respiratory centres in CNS

There are two main respiratory centres in the central nervous system. They are the pons respiratory centre and the medulla respiratory centre, named after the area where they are found in the brain. The pons respiratory centre can be subdivided into the apneustic centre, which increases the depth of breathing, and the pneumotaxic centre, which decreases depth of breathing. (Note: in PHYL3002 our lecturer just told us that the apneustic centre might not actually exist 0_o) The medullary respiratory centre can be broken down into the dorsal respiratory group (DRG) and ventral respiratory group (VRG). Directly superior to the VRG is the Pre-Bötzinger complex, which is like a pacemaker for the respiratory system. I'll go into more detail on each of these areas throughout the post.

Discuss the generation of the basic rhythm

As I just mentioned, the Pre-Bötzinger complex is the pacemaker and is thus largely responsible for the generation of the rhythm of breathing. It sends signals to the dorsal respiratory group, which causes the diaphragm to contract. These signals don't come all at once- they gradually increase in intensity as an "inspiratory ramp signal." If this didn't happen then all of our breaths would be short and sharp, and we wouldn't be able to do things that require you to control your ventilation, such as talk or play clarinet. Boo.

The DRG is close to the VRG, so when very strong signals are sent to the DRG, they may "spill over" into the VRG. This causes other "accessory" muscles such as the rectus abdominus and internal intercostals to aid in forced expiration. (For more information on muscles involved, please see this post for ANHB2212.)

The pneumotaxic and apneustic centres also influence our breathing. The pneumotaxic centre, as mentioned before, limits our breathing- I remember it as kicking in when breathing is too taxing (pneumotaxic). It can "switch off" the signalling between the DRG and inspiratory muscles. The apneustic centre, however, stops the signal from being "switched off." Normally the pneumotaxic centre dominates, but if the apneustic centre dominates, such as in a condition known as apneusis, the patient may breathe by long inspiratory gasps interrupted by short, brief expirations. (Note: As I mentioned above, one of my lecturers for PHYL3002 has disupted the existence of the apneustic centre.)

Discuss the control of ventilation by P_O2 & P_CO2

When we breathe in, our P_O2 increases and P_CO2 decreases. The opposite is true for when we hold our breath. These differences are the basis of control of ventilation. Changes in our P_O2 and P_CO2 are detected by central and peripheral chemoreceptors.

An increase in P_CO2, as detected by the central chemoreceptors in the medulla, is the main stimulus for increasing our breathing rate. CO₂ can cross the blood brain barrier, where it interacts with water to form H⁺ and HCO₃^-. The H⁺ can be detected by the central chemoreceptors, which then send signals to the respiratory control centres, increasing ventilation. This only occurs up to a point, however: at extremely high P_CO2 levels ( > 70-80 mmHg), the entire brain gets depressed, including the respiratory neurons. Not good.

A decrease in P_O2, as detected by peripheral chemoreceptors in the carotid arteries and aortic arch, can also stimulate an increase in breathing rate. This only happens, however, once P_O2 drops below around 60mmHg, which is pretty rare since normally a rise in P_CO2 would cause us to breathe before this happens. Just like P_CO2, however, if P_O2 drops too low, then the entire brain gets depressed and can't breathe. Another trap to watch out for is that P_O2 is only a measure of dissolved oxygen, and not the total amount (i.e. dissolved oxygen plus oxygen attached to haemoglobin). Hence, in severe anaemia or in carbon monoxide poisoning (where carbon monoxide preferentially binds to haemoglobin rather than oxygen), this respiratory reflex will not kick in and so the patient might die of oxygen deprivation.

Understand exercise induced hyperventilation 

As I'm sure you are well aware, you breathe faster when you exercise. This isn't so much due to changes in P_CO2 or P_O2- during heavy exercise, sometimes our breathing rate is so great that P_CO2 might actually decline or P_O2 might increase. Also, breathing rate often increases before exercise, as a kind of anticipatory response.

Here are some possible mechanisms through which exercise increases ventilation:

• Input from higher cortical centres- this might explain why breathing rate increases before exercise.
• Proprioception- input from receptors in our muscles etc.
• Increase in body temperature
• Secretion of stress hormones such as adrenaline
• Increased sensitivity of chemoreceptors

Now for a quickie on how breathing rates and CO₂ levels fluctuate during and after exercise! As I just mentioned, when you start exercising, or slightly before you start exercising, your breathing rate increases. As your metabolic rate hasn't actually increased yet, you will be blowing off more CO₂ than you are producing, so your CO₂ levels will drop temporarily. As you exercise, your CO₂ production will increase, so eventually your CO₂ levels will be back to normal. When you finish exercising, your breathing will return to normal, but it will be a bit longer before your metabolic rate decreases as well. Hence your CO₂ levels will increase temporarily following exercise.

Discuss other reflexes controlling respiration

There are two main reflexes that you need to know:

1. Hering-Breuer Inflation Reflex: This reflex prevents overinflation of the lungs. Overinflation is picked up by stretch receptors in the smooth muscle of the airways.
2. Irritant Receptor Reflex: This is essentially just coughing or sneezing to get rid of irritants.

And that's it for respiration! Next I'll go back to talking about cardiovascular physiology (which was actually meant to be the first topic after control systems, but I was having issues with the respiratory stuff *cough*Flow-Volume plots*cough*).

Antibodies as Tools of Discovery

Final post for biochemistry!

Definitions

First of all I'm going to start with some definitions for you:

• Antigen- A foreign substance that elicits an immune response.
• Antibody- An immunoglobulin protein that binds to an antigen.
• Epitope- A part of the antigen where an antibody binds.
• Polyclonal antibodies- A mixture of different antibodies. Different antibodies may bind to different epitopes on the same antigen.
• Hapten- A small molecule that doesn't elicit an immune response by itself, though it may elicit an immune response when bound to some other protein. Some of the antibodies raised against a hapten-protein complex may also bind to the hapten on its own.

Immunoglobulin G (IgG)

As I mentioned before, antibodies are immunoglobulin proteins. There are five types of immunoglobulin proteins, but for now we're only going to focus on one: IgG.

IgG is kind of Y-shaped. It is made up of two heavy chains, as well as two light chains inside the two small arms of the Y. The light and heavy chains are bound to each other by disulfide bonds, and the heavy chains are also bound to each other by disulfide bonds. Additionally, there are intrachain disulfide bonds that afford greater stability of the protein. Each L-chain consists of two Ig-fold domains, whereas each H-chain consists of four Ig-fold domains. These Ig-fold domains are simply beta barrels (see my earlier post for more information on how proteins fold).

There are many different IgGs that can attack different epitopes on different antigens. All IgGs have a constant sequence, taking up the long arm of the Y as well as half of each of the small arms. The tips of the small arms of the antibodies, where antigens bind, are variable sequences. Antigens bind to these areas quite tightly, with a dissociation constant usually less than 10^-9 M (that's really small).

It is possible to use the protease papain to cleave IgG just below the fork of the Y. This creates two F_ab fragments, each of which can still bind an antigen. This is useful when you want small antigen-binding molecules.

Creating Diverse Antibodies

There are millions of antibodies, so it wouldn't be very efficient if there were different genes coding for every single antibody. Thankfully, as I mentioned above, IgGs all have constant sequences, which is rather helpful.

First, let's look at light chains, or more specifically at κ-type light chains, which are the main light chains in humans and mice. In the genome, there is an "L_κ + V_κ Library," which contains pairs of L_κ (leader/Targeting sequences- ensure that the mRNA is translated on the endoplamic reticulum) and V_κ (variable region coding sequences). These are then spliced with one of five J_κ (joining sequences) and finally with the one and only C_κ (constant region coding sequence). There are roughly 100 L_κ/V_κ pairs, and five J_κs, resulting in over 500 combinations (additional combinations are created by random deletions when things get spliced together).

Heavy chains are pretty similar, but instead of the sequences being called L_κ, V_κ etc., they're called L_H, V_H etc. There is also one extra step: L_H/V_H sequences are spliced with one of 30 D_H (diversity sequences), then to one of six J_Hs, and finally to C_H. Random addition or deletion of nucleotides can create even more combinations.

Note that I only talked about variations within chains. There is even more variation when you take into account that different light chains can pair up with different heavy chains, and so on and so forth.

Antibody Production

To use antibodies as effective tools, we need a way to produce them. Here's how:

• An animal (e.g. mouse) is injected with an antigen, and spleen cells, some of which are making antibodies to the antigen, are extracted
• These spleen cells are fused with mutant mouse myeloma cells that are unable to grow on a medium called HAT. This results in the production of three kinds of cells:
  1. Unfused spleen cells that die not long after being out of the animal
  2. Unfused myeloma cells that die when on HAT
  3. Fused cells that produce antibodies and don't die
• Single cells are cultured in separate well. Each cell produces monoclonal antibodies but doesn't die

Uses for Antibodies

There are plenty of uses for antibodies, one of which (ELISA) you have already seen. Here are some other uses:

• Western Blotting- kinda like Northern/Southern blotting but for proteins. Antibodies are used to detect the protein of interest.
• Immunocytochemistry- using fluorescently tagged antibodies to detect proteins within the cell. Requires cells to be fixed and pores created in the cell membrane.
• Drug targeting- Antibodies against a cell surface marker can be coupled with cytotoxic or radioactive reagents- helpful for killing off cancerous cells.

Proteolytic Enzymes

This post involves a love story too, though it's a somewhat different love story.

Meet Nucleophile and Electrophile. Nucleophiles (nucleus-lovers) tend to be attracted to positively-charged things (generally stuff with relatively few electrons), and electrophiles (electron-lovers) tend to be attracted to negatively-charged things (stuff with relatively more electrons):

(Sorry if that was a shitty and/or inaccurate description btw, I don't know how to explain this.)

Occasionally nucleophiles impose themselves (for lack of a better word) on the electrophiles. This process is known as "nucleophilic attack" and is much less brutal than it sounds. A lot of reactions involve such nucleophilic attacks. These include reactions by proteolytic enzymes that cleave peptide bonds.

One type of catalytic reaction is acid catalysis. This is relatively rare in biological systems as it requires very low pHs, and generally biological systems are at a pH of around 7. It's a fairly good place to start though, so let's take a look at how acid catalysis works.

In peptide bonds, there is a carboxyl group and an amino group joined together. The oxygen on the carboxyl group (the carbonyl oxygen) tends to carry a slight negative charge as O is more electronegative (more likely to attract electrons) than C. This makes the O nucleophilic. Hydrogen ions floating around in acid solution are electrophilic- they're just a proton, after all. Hence the carbonyl oxygen attacks the H⁺, sharing some electrons with it, like so:

This results in the breakage of one of the C-O bonds and the formation of an O-H bond. The removal of electrons from the C=O bond also makes the carbon less negatively charged (more positively charged) so that water can come in and attack. At the same time, more crazy attacking stuff happens: namely, the amide group attacks one of the hydrogen atoms on the water, and the C=O bond reforms:

This results in the formation of separate carboxylic acid and amino groups, like so:

Note that the H+ did not change in the reaction. Remember, catalysts speed things up without actually being changed or consumed by the reaction.

General acid catalysis is done using weak acids, rather than strong acids, so general acid catalysis is more likely to be seen in biological systems. The process is pretty similar to acid catalysis, except that the water nucleophilically attacks the carbonyl carbon at the same time that the weak acid protonates the carbonyl oxygen. (Sorry, no pictures, I'm done drawing already.) When the water nucleophilically attacks the carbonyl carbon, the O and one H of the water bind, leaving another H around. This H gets nucleophilically attacked by the amino group, and the C=O reforms.

Metal ion catalysis is similar to general acid catalysis, but with a metal ion rather than a weak acid.

Hydroxide ion catalysis is the opposite of acid catalysis: it occurs at very high pHs. Here you have hydroxide ions floating around in solution. This is kinda similar to the other three above, except no protonation or addition of a metal ion is needed at the beginning. Instead, a hydroxide group directly attacks the carbonyl carbon, and one of the C-O bond breaks to leave the carbonyl oxygen negatively charged. Then the amino group attacks one of the hydrogens on the water, and the C=O reforms (sound familiar)? The loss of the one hydrogen from the water means that the original OH^- group is reformed as well.

Finally, in general base catalysis, the weak base attacks one of the hydrogens on water (forming a weak base bonded to H), and the resulting OH^- group attacks the carbonyl carbon. This results in the breaking of one of the C-O bonds to leave the carbonyl oxygen negatively charged (just like in hydroxide ion catalysis). The amino group then attacks the H bonded to the weak base, and the C=O reforms.

Overall, these catalysis reactions can be summarised in three main steps:

1. Something nucleophilically attacks something else
2. A tetrahedral transition state forms
3. The C-N bond breaks by the amide group nucleophilically attacking a hydrogen, and the C=O bond reforms.

Now, why go on and on about catalysis? That's because proteolytic enzymes use these reactions to catalyse the breakage of bonds.

Proteolytic Enzymes

There are four main kinds of proteolytic enzymes, generally named after the residues at the active site (NOT the places where they cause breakages).

1. Serine proteases- contain serine in the active site
2. Thiol proteases- contain cysteine in the active site
3. Metalloproteases- contain a metal ion in the active site
4. Acid (Aspartate) proteases- operate at acidic pHs, contain two essential aspartate residues in the active site

Serine proteases

Serine proteases contain a "catalytic triad" of three essential residues: serine, histidine and aspartic acid (aspartate). The first steps involve general base catalysis followed by general acid catalysis: the histidine acts as a general base allowing the -OH group on serine to nucleophilically attack the carbonyl carbon. After the breakage of the C-N bond, the carbonyl group is still attached to the serine, whereas the amino group is released from histidine (histidine donates an H, like an acid). This allows histidine to then act as a general base again, stealing an H from water to allow the -OH to attack the carbonyl carbon and release the carboxyl group from serine. (The H that histidine stole gets transferred back to serine- again, kinda like an acid.)

As for the aspartate group? That doesn't directly contribute, but its negative charge at biological pH may help stabilise histidine as it becomes more positively charged during parts of the process.

There are three main serine proteases that you should know: trypsin, chymotrypsin and elastase. Trypsin breaks bonds after lysine and arginine. Chymotrypsin breaks bonds after large, bulky amino acids like tryptophan and tyrosine whereas elastase breaks bonds after little things like alanine and valine. (This is by no means a comprehensive list.)

Thiol proteases

Thiol proteases act in a similar way to serine proteases, but instead of a catalytic triad, they have a "catalytic diad." This diad includes histidine and cysteine. Thiol proteases such as papain have a broad specificity. They do have some preferences, but they have so many preferences that they might as well not have preferences- kinda like people who prefer to drink on all days ending in Y.

Metalloproteases

Metalloproteases seem to act mainly by metal ion catalysis: the water nucleophilically attacks the carbonyl carbon, helped out by the metal ion, which helps to stabilise the negatively-charged carbonyl oxygen in the transition state. Glutamate, which deprotonates water, helps out too. A tyrosine residue may also play a role in breaking the bond, but this is unclear.

Metalloproteases tend not to have specificity for any particular amino acid- they tend to simply hydrolyse the last bond in the chain.

Acid (Aspartate) proteases

As their name suggests, acid proteases work best at acidic pHs. They have two essential aspartate residues. One of these is usually deprotonated, while the other is protonated. The deprotonated aspartate residue acts as a general base to help water attack, whereas the protonated aspartate residue acts as a general acid to make the carbonyl carbon more positively charged, thus helping along the process. This means that the originally protonated aspartate is now deprotonated, and vice versa. The aspartate residues are close enough for this to reverse, and then more general base/general acid action can occur to allow the bond to break.

Acid proteases tend to catalyse bonds between aromatic amino acids, such as phenylalanine and tyrosine.

One example of an acid protease that you should know is HIV Protease. I've written about it as well as drugs that target it in a previous post for PHAR2210.

Enzymes as Tools of Discovery

This was probably the most interesting lecture in the Enzyme Kinetics module (that we've had so far, that is- our last lecture's tomorrow). Let's see what it entails!

Ribozymes

Ribozymes are enzymes that are made out of RNA (rather than protein). They usually catalyse reactions involved in the synthesis and processing of other RNAs. For example ribozymes, helped along by guanosine, catalyse the removal of introns in the processing of Tetrahymena rRNA. (As far as I know we don't have to know the mechanism in detail, so let's not get bogged down in the details.)

Abzymes

Abzymes are basically enzymes that are designed to catalyse particular reactions. Abzyme design is based on two main principles: that enzymes catalyse reactions by binding the transition state very tightly, and that antibodies bind things very tightly. Hence, the idea here is to make antibodies against transition states.

Abzyme creation isn't too hard to understand: first you choose a molecule that is very similar to the transition state (you can't choose the transition state itself as it's very unstable) and then you raise antibodies against that transition state. How those antibodies are raised isn't something that we need to know for now, but it'll probably come up if you're going forth in Biochemistry.

Enzymes as Tools

Being able to design enzymes is all very well and good, but that probably wouldn't be so useful if we couldn't use enzymes as tools! Enzymes, both natural and designed, can be useful tools thanks to their specificity and their ability to catalyse a lot of reactions so that only a small amount of enzyme is needed.

Here are some examples of where enzymes can be used as tools:

• Restriction enzymes- used to cut DNA.
• Specific proteases- used to cut proteins.
• PCR- used to amplify DNA segments.
• ELISA (Enzyme-Linked ImmunoSorbent Assay)- uses a small amount of enzyme to catalyse the formation of a large amount of product, thereby allowing small amounts of antigen to be detected.
• Markers for cloning- see my earlier post to see how cloning and enzyme activity/inactivity can be used to see whether a plasmid has taken up a vector or not.

I'm not going to talk about restriction enzymes or PCR, as they've been done to death on this blog. I also won't go into any more detail about cloning markers, as this lecture didn't go beyond what I mentioned on that earlier post.

As for proteases, I won't go into much detail on the proteases themselves (the next post will be about proteases) but what I will talk about is a funky 2D gel on that slide that can be used to separate out proteins. The reason it's called a 2D gel is because proteins on it are separated out according to two characteristics: size and pKa. Along one side of the gel there is a pH gradient, and proteins will settle at the pH that matches their isoelectric point (i.e. when they're neutrally charged- see my earlier post for a bit more information about isoelectric points). Perpendicular to this gradient, a charge is run across the gel, just like in agarose gel electrophoresis. This separates proteins out according to their size.

After this is done, you can cut out a part of the gel containing the protein that you're interested in. From there, you can use proteases to cut the protein, and mass spectrometry to find the size of the fragments. You can then use this information to search databases to find out what protein you're looking at.

Now I'll talk a little bit about ELISA and give an example of how it's used to detect HIV!

The first step in testing for HIV is to make some rabbit antibodies against it. This involves giving rabbits some of the HIV coat proteins- not the whole virus, just the coat. The rabbits can then raise IgG against it.

Some more important molecules include horseradish peroxidase and goat IgG. Horseradish peroxidase can bind to goat IgG. You'll see why this is important in a bit.

Samples containing HIV can be put into a polystyrene dish, with rabbit IgG added. This is then washed to remove unbound antibodies. The horseradish peroxidase/goat IgG complex can then bind to rabbit IgG (yup, they can all bind together, how convenient). The horseradish peroxidase can then do its work in converting colourless substrates into coloured products (unfortunately, I don't know what substrates/products are involved in this reaction). The amount of coloured product formed is proportional to the amount of peroxidase present.

What is Pharmacology?

During the revision lecture, the unit coordinator told us not to assume that there wouldn't be a short-answer question on Lecture 1. And then I realised that I hadn't blogged about it. I thought I was done blogging about pharmacology, but apparently not!

Define the term “pharmacology” & the scope of the discipline

Pharmacology is essentially the study of how drugs and other biologically active substances affect the body. We don't worry so much about how drugs and so forth are actually prescribed, that's pharmacy's job.

Show awareness of the importance of plant-based medicines to the historical emergence of pharmacology, identifying 3 drugs that came from botanical sources

Originally we used plants as medicine due to the presence of many bioactive compounds.

One of the most well-known examples of a modern drug that originally came from a plant is aspirin. Aspirin originally came from willow bark, used as far back as ~200AD in Ancient Rome. In 1828 Büchner isolated salicin, the active component of willow bark. In 1897, aspirin, or acetylated salicylic acid, was patented and synthesised by the drug and dye firm Bayer.

A lesser-known example of a drug that came from a plant is atropine. Atropine originally came from deadly nightshade, a.k.a. belladonna. "Belladonna" actually means "beautiful woman," and the name came about because it can cause rosy cheeks and dilated pupils, which people thought was beautiful. (It does eventually cause death though, so there's that.) In 1831 Mein isolated atropine from belladonna, and in 1867 it was found to block cardiac vagal stimulation. (Atropine blocks muscarinic cholinergic receptors, as mentioned in my post about the autonomic nervous system.)

A third example is artemisinin, used to treat malaria. In 340AD, Sweet Annie (Artemisia annua) was used by Ge Hong to alleviate fever. In 1596 another Chinese herbalist, Li Shizhen, reported using it to treat malarial fever. It wasn't until the 1970s that the active ingredient (artemisinin) was isolated, however. One of the scientists working on this discovery, Tu Youyou, won a Nobel Prize for her work.

Appreciate how rising awareness of the limitations of plant-based medicines drove the growth of modern pharmacology

Plants are great, but they aren't perfect. For example, it's very hard to determine how much of an active ingredient you have in a plant. There may also be other compounds in the plant that might interfere with the activity of the active ingredient. Combined, these make it difficult to test drugs and determine a safe dose.

Another limitation of using plants is that you're limited to what you can find in nature. With technology, you can tweak molecules around to make drugs that are safer and/or more effective. So next time someone comes up to you saying "why take nasty synthetic aspirin when you can just chew on some lovely natural willow bark?" you've got a few things that you can yell right back at them.

Understand how 3 different classes of names are commonly assigned to drugs.

Drugs have at least three different names. They have a chemical name, a generic name and one or more brand names.

The chemical name of a drug is simply the name given by IUPAC. These are extremely descriptive, but are often quite a mouthful. For example, if you have depression you might be prescribed some (S)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile. Have fun asking the chemist for a refill of your (S)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile, though.

The generic name is a much simpler name given to the drug molecule that sounds somewhat sophisticated and classy, but not catchy enough to be a brand name. Normally they have prefixes or suffixes that are related to their function- for example, all of the penicillins end with -cillin. Just in case you were curious, (S)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile is also known as escitalopram. I still have no idea if I'm pronouncing it correctly (I've heard three different pronunciations of that name, two from the same person), but at least it's a lot easier to pronounce than the chemical name.

Finally, drugs have a brand name that sounds kinda catchy so that it sticks in people's minds. For example, paracetamol is often sold as Panadol, and escitalopram is often sold as Lexapro. After the drug is off its patent, other companies can sell the same drug under different brand names. For example, Lexapro is also sold under the brand name Lexam (they kept the first four letters the same, how original).

Show a basic appreciation of the importance of “pharmacokinetics” and “pharmacodymanics” to understanding the effects of drugs on the human body.

Pharmacodynamics is what a drug does to the body, and pharmacokinetics is what the body does to the drug. For more information, just read all of my other pharmacology posts :P