Biology 1A - Lecture 31: Endocrine System

Uploaded by UCBerkeley on 09.11.2012

>>INSTRUCTOR: Good morning. So, let's get started.
I hope you've got your boots on, we're proceeding further into the immune system today. When
last we met we were talking about the anti microbial peptides in the Drosophilaハ sorry,
the Drosophila system, and we were, you, you know, dealing with this question, whether
a single peptide could be sufficient to account for the anti microbial effect or maybe in
other okays, multiple peptides might be required to get full potency.
Another question that arises is, you know, that we'll come back to several times, this
question of self verses nonself, how do these peptidesハ the peptide level, how do these
peptides distinguish foreign cells from the host cells in the innate immune response?
And remember, we mentioned the central feature in the innate immune response is that the
guard cells, the defensive cells are recognizing molecular patterns associated with, you know,
pathogens or microbes, generally, so there's these PAMPs or MAMPs and here I'llハ this
provides an example of this phenomenon. That it turns out that bacterial plasma membranes
were enriched for negatively charged. Phospholipids on they're outer face and on the inner face
of the membrane, they have neutral lipids. So if you're talking about bacteria, here
are the two lipid bilayer and it's not neutral lipids inside, and enrichment for negative
lipids outside, where as in plant and animal cells, there are mainlyハ so give these guys
a nucleus now, these types of cells have negative charge lipids on the inside and mostly neutral
on the outハside. Okay.
And this sort of global difference between the two classes of cells, allows you to devise
a molecular strategy for attacking the bacteria selectively with secreted peptides.
And it turns out, when people looked at a large number of anti microbial peptides from
a large number of species you look for Drosophila, human, plants, whatever, theyハ it was realized
that although the amino acids amino acid sequences are wildly variable, these peptides have certain
features in common is that they're mainly a mixtureハ they're amphipathic featuring
mainly cationic or neutral domains, okay of amino acids. And sometimes these are there
might be a sequence that looks like it's not the positively charged amino acids scattered
along the peptide sequence, but when you think about how this might fold up into an alpha
helix and then again you see that the positively charged amino acids are in one side and neutral
amino acids on the other. So these are amphipatic molecules.
Okay. Sort of like a detergent. And what happens
then is that if these peptides interact with eukaryotic cells, it's going be sort of a
weak hydrophobic interaction between the peptideハ hydrophobic domain and the outer leaflets,
largely neutral in these eukaryotic cells. In contrast the anti microbial peptide positive
domains can interact very strongly by electrostatic interactions with the outer leaflet of the
bacterial cell membrane. And that allows these things then to intercalate(?)
into the surface and that actually increases the surface area, when you're putting these
molecules into the outer leaflet, that expands the outer leaflet relative to the inner leaflet
and starts giving disruptions so that eventually pores can form that allow the nonselective
influx and efflux of molecules and ions from the cell and these can either then break up
or sometimes the peptides also go in and interact with other molecules inside the cells.
Okay. So, the difference in the composition of the
bacterial cell membrane verses eukaryotic membranes allows you to have a self/non self
sort of crude distinction made. Questions about this?
So let's go back to this question of theハ the fact that the innate immune system functionally,
all sorts of animals and plants can guard against this sort of reaction, infections
by bacteria, with innate immunity and when you look at the pathways that are involved
in mammals and even in plants, you see that the protein receptors ton surface areハ have
structural similarities, genetic similarity to the receptors that are seen in Drosophila,
that they had a common evolutionary ancestry. In addition, some of the signal transduction
molecules, such as kinases or kinase activity, and then the final kinase cascade that leads
to the activation of some cellular response. Those are also similar, they're homologous
genes and proteins across this side evolutionary span so the conclusion from that is probably
the innate immune system was there inハ haハ has been there for a long long time, even
before there were multicellular animals and plants you have to think about when these
things evolved. But the idea is that, anyway, that these could
be ancient process, innate immunity. In the vertebrate system, there's been a duplication
of these receptor proteins that we may hear about again toll like receptors, where in
Drosophila, there's only one or two, there areハ I don't know, 5 orハ10 in mammals
and they have evolved different lee gun specificities so some are receptiveハ that are activated
by lipopolysaccharides like from bacteria cell walls, others are activated by binding
to the flagellar proteins, flagella is a protein in the swimming business of invaders, okay.
Others are activated by double stranded RNA. Here again is a different version of a molecular
microbial associated pathogen, molecular pattern the pathogen, use eukaryotic cells don't have
much double stranded RNA this is usually an indication that you've been attacked by a
virus of some sort. Retrovirus. Similarly, you can have DNA where you have
CpG repeats, cytosine, guanine, repeats in eukaryotic cells, most of these parts of DNA
are often modified by methylation, and in bacteria they're not.
So, if you have CpG repeats that are not methylated, again that can be sort of a clue that all
is not right in your cells. Okay.
You'll notice that these nucleic acid receptors are not found on the outer surface of the
cells but rather vesicles within the organism. Within the cell.
And we'llハ um, sorry. Just listen to that because I hear an iClicker question approaching.
So, what happens is that, you know, in general, these phagocytic cells that are like macrophages
that are patrolling the host animal they have these various receptors and theyハ when they
sense a pathogen, that initiates a cellular response of phagocytosis they send out arms
of the plasma membrane that develop this and seal off and bring this pathogen inside of
the cell within a vacuole. Right. And then these vacuoles, this is just a regular
endocytic pathway, pretty much, they can use with previously existing intracellular vacuole
that is a lysosome that has all of sorts of digestive enzymes in it and other toxic molecule
producers and when these two fuse and then you can sort to degrade this pathogen. Okay.
So, here's the iClicker question. Why would the toll like receptors that bind
DNA and double stranded RNA, why are they in the vesicles of the endocytic pathway?
Okay. Rather than the outside of the cell. It could be because toll like receptor proteins
generate free amino acids for histamine biosynthesis. That doesn't even make sense to me. But anyway.
Sorry. But don't let that bias your answer it could still be correct.
This observation is probably an artifact. Because toll like receptors only function
at the plasma membrane. Binding of nucleic acid receptors is so slow that the binding
onlyハoccurs when the receptor in the ligand are confined in a small place for a long time.
D. pathogen nucleic acid are protected by viral coat protein and are only expose add
the pathogens are degraded within the lysosome or all of the above.
We'll give youハ cow you shouldn't need more than 3 seconds but I'll give you 15ハ I'll
give you 20, go I can turn the darn thing on.
Okay. 10 more seconds.
Okay. Right.
I don't think we need to discuss this further. Right.
So, sort of take itハ yes? Thereユs a question. >>STUDENT: [Indiscernible]
>>INSTRUCTOR: Theハ okay. So, are the toll like receptors, originally
in the plasma membrane and come in. I want to say that the toll like receptors
do not break down anything, they just say that it's there.
And they initiate a response that eventually leadsハ okay.
>>STUDENT: [Indiscernible] >>INSTRUCTOR: Theyハ soハ so theハ the
receptors are just receptors. Whether it's a nucleic acids receptor or a lipopolysaccharide
receptor or a flagellin receptor it just says, ah, there's flagellin here and that activates
that molecule and then stuff downstream initiates the cellular response. Okay. That was your
first part of the question. Or something I wanted to clarify.
Second, my understanding is that the toll like receptors that are specific for nucleic
acids reside in theハ ha, they're mainly in these vacuoles, now how do they get in
the vacuoles if they're not initiallyハ that's your question and the answer is, I do not
know. But they functionハ they might be inserted
later, there might beハ they might fuse with other vesicles into theハ into the primary
endosome that's a good question. So, again, taking sort of a high view, innate
immunity in mammals we have the barrier defenses. And in your layers of skin, as they arise
and then the skin cells die and differentiate while they're dying into a very carefully
structured stuff that sheds off everyハ so every 28 days you're a new person in the mirror,
I just learned that fact. So, theseハ thisハ these layers of cells
are sort of brick wall of dead cells, is infused with antimicrobial peptides so that's the
outer stuff, but the linings of your respiratory system and feeding tubes are also technically
outside, so there you've got mucus membranes lining it and so there you can have living
cells sort of patrolling within the mucus for invading cells.
So, we talked about mainly the cellular defensesハ again this is innate immunity, cellular defenses
include various sorts of cells that can phagocytize invaders there are neutrophils in the blood
these are attracted to sites of infection or damage, by chemical signals from the original
insult. And then there are macrophages that are sort
of wondering through out the body or resident in spots, such as the spleen and the lymph
system where they're more likely to encounter invaders. Okay.
And theseハ and then there are dendritic cells that are mostly seen near the environment,
like the skin, so they're ready to interact with foreign cells.
And these macrophages and dendritic cells are important later because they also involveハ
or involved in the adaptive immune system. And then the cells that are patrolling outside,
in the mucosal environment are typically cells called: Eosハ yeah.
That's misspelled. Eosinophils.
Eosine is a dye that's used to stain cells and the cells that stain well with eosine
that are within eosin are eosinophiles. So that's a misspelling.
So here's a picture of a macrophage. Macrophage means big eater.
So, these are large cells and theyハ here's one that's engulfing a yeast. In contrast
the dendritic cells, dendriticハ dendrites branches, okay, so they're structures are
highly branched, they're not really yellow, these are pseudo colored or in this case florescence
image, so these are highly branched cells that can reach out and grab someone.
Okay. Just examples, diverse morphology.
And then there's a third type of cell that we'll hear about later, natural killer cells,
so these phagocytic cells are mainly engulfing cells, foreign cells. But let's say you have
a viral infection or an intracellular bacteria, like listeria, then theハ those guys might
get inside of the cell and think, home free, but not really, because you have these circulating
cells called natural killer cells that recognize when something is going wrong on the host
cells. Okay.
There's a change in the surface biochemistry of host cells, frequently, when they're undergoing,
you know, when they're involved in cancer when they're involvedハ invading by a virus
or neutracellular bacteria, but when they sense the abnormality they will kill host
cells that are expressing abnormal proteins on their surface. Okay.
So, this innate immunity itself is quite a complicatedハ the molecular aspects of the
defenses include antimicrobial peptides, various secreted signals proteins that may activate
neighboring cells to block viral reproductions or stimulate the macrophages to increase phagocytosis,
there's also protein complex called: Complement, this is 25 or 30 different proteins that are
produced and circulating in the plasma and interstitial fluid, okay.
And thisハ it's sort of like blood clotting, a complicated biochemical cascade, but now
involving a different set of proteins and the activation of this complex is by infection,
and that can activate to form complexes that, again, sort of disrupt membranes and lice
invading cells and that trigger the inflammatory or participate in the inflammatory response
which we'll get to in a bit. Finally, another thing that happens is that
in theハ another way that a macrophage might kill cells, once it's engulfed the cell, and
fuses with a lysosome there are enzymes in here that generate toxic small molecules,
highly reactive, they're called generically reactive oxygen species, reactantハ reactive
nitrogen species. Again, you don't need to know this for the
rest, but just an example, you can take the amino acid arginine and act on it enzymatically
to produce nitric oxide a highly reactive free radical, so when they say highly reactive,
it means it's going to ore act element instantaneously, whatever it meads meets first in the diffusion
so they have a short path length, and they screw up the proteins and help kill them.
Kill the organism. Okay.
Oh, my gosh. So, allow do you think this specificity of
reactive oxygen and reactive nitrogen species, how do theyハ how do they know to kill the
invading cell and not the host cell? Well, it could be that these small molecules,
such as nitric oxide and superoxide only recognize pathogen-derived molecules.
Or they're produced locally within the cell and are so highly reactive that they cannot
diffuse far from the site of production, I will give you 15 seconds that's ridiculous.
It's an insult to your intelligence. 15 seconds.
Go. Go. Go. Go. Go. Hurry, hurry, hurry. Okay.
You guys are taking advantage of my slow reflex but that's okay.
The answer is ooh. Okay. Well, you've got to focus, you to keep payingハ
I was just talking how these things canハ they can only diffuse, you know, in an means
they're going to react instantly they're produced locally within the cell, so what they're going
to encounter first is that those bacterial proteins.
Now, they do eventuallyハ it's hard work being a macrophage, because you're constantly
battling these foreign cells and you are constantly producing these toxic molecules every time
you try to swallow one, so these guys can typically do about 100 cycles of engulfment
and degradation before they wear themselves out.
Okay. Whoops.
Yes? >>STUDENT: [Indiscernible]
>>INSTRUCTOR: The answer was B. It's highly he reactive diffusing a short
distance so there's noハ how could it be that something like O ハcould distinguish
between a host cell and any other cell, yeah? >>STUDENT: [Indiscernible]
>>INSTRUCTOR: Ah, are is what foundedハ the reactive oxygen and nitrogen species, they're
used by they're deployment by macrophages, I know that remarkably that nitric oxideハis
also used as a neurotransmitter, we may get to that, so it's not onlyハ and it acts on
smooth muscle, also, I thinkハ so, these are common signalsハ they're surprisingly
widely used as signaling molecules. We already did that, right?
So just to look theハ the lymphatic system is highlyハ heavily involved in immunity.
Remember the lymphatic system is basically, the slowly flowing interstitial fluid. Right.
Plasma leaks out of the capillaries, due to the difference between the blood pressure
and the osmotic pressure, so you have a slow leak of plasma proteins from blood into interstitial
fluid that has toハ in order to close the circuit, that material flows then through
all of the different cells so it's bathing all of the cells intimately, gradually collecting
into these ducts and reentering the circulatory system at the veins in the neck.
Okay. Where the backpressure would be the lowest.
There are various lymph nodes all through the system.
And at these lymph nodes, it's sort of like the neighborhood bar, whatever, where all
of the immune cells macrophages and other immune system cells hang out because pretty
much everybody is going to go through there and then they can use these surveillance sites,
sort of like a roadblock to look for invading cells.
Okay. And that's why doctors are interested when
you have an inflamed lymph node or looking at metastasis in cancer they will look for
the lymph nodes to look for cells that are out there where they shouldn't be.
So, final comment on the innate immune response is there's something called the inflammatory
response, I'm sure you've all had it when you've got a splinter or a localized infection.
The macrophages are there on the job, and they'll secreteハ in addition to carrying
out they're phagocytosis they secrete signals molecules, cytokines that are variety of functions,
first they'll increase the permeability, the leakiness of the capillary near by. Making
it easier for fluid to flow outハand neutrophils are drawn by chemotaxis and they migrate out
through the endothelial wall to the site of infection and help in the phagocytosis they're
also triggering release of histamines and by mass cells that increase blood flow so
you get redness and increase in temperature, because you've got more blood flowing in.
And theseハ with luck these things participate in cleaning up that wound and then things
go back to normal. Okay.
And the complement system is also involved, as part of this sort of complex inflammatory
response. And I'm sure you've also seen this, this accumulation
of fluid and local heating, all of the deadハ the dead cell debris the worn out macrophages
you get this sort of accumulation of pus, so actually it's not bad it's just a sign
that things are pretty much working. Questions about innate immunity? Yes?
>>STUDENT: [Indiscernible] >>INSTRUCTOR: Okay.
What makes interstitial fluid flow? Okay.
Well, basically, it's theハ where interstitial fluid comes out from the capillaries, it's
being driven out by the blood pressure in the capillaries, which is low, but still finite,
okay. And where the interstitial fluid is reentering
the circulation, that's in the veins right next to the heart, the big veins right next
to the heart and the pressure there is even lower, okay.
So, you do have a pressure differential between the site of origin of the interstitial fluid
and where it flows in. So that slow pressure differential gives you a net flow. Okay.
And I think you probably seen this if you get a big bruise in your leg or something,
you get all sorts of local breaking of blood vessels and there's all sorts of crud that
go out into the blood vessels and the interstitial fluid, you will see the bruise is here, but
a week later, the green, yellow and red is moving down your leg, right?
By force of gravity in the net flow of the interstitial fluid and then it gets taken
into the lymph vessels and then gets back up here and clears out byハ is that the liver?
We haven't got there yet. It's nice to be in the hands of a real expert,
right? So, adaptive immunity.
Remember this is the immunity that is unique to vertebrates. Okay.
And it's been noticed antidotally for a long time, there's this guy writing in Athens,
during the wars with some other Greek town, all of the people from the surrounding villages
had come into the big city to be protected by the Athenian navy, what do you call it
hygiene broke down as a result. Probably problems and there was some disease, perhaps typhoid
fever that ended up killing 1/3 of the population. There was a severe plague that hit them, but
this guy, Thucydides, noticed that those who survived the third bout of the disease were
able to care for others without being re infected. So that's this adaptive immunity.
And so, theハ there's some remarkable features of this is that we'll see that there's this
tremendous diversity that you're immune system can detect and react and fight off molecules,
pathogens, that it's never seen before. Okay.
And it can react to millions of different molecules, okay, including, you know, synthetic
molecules that have never occurred in nature. Right.
How can that be? Also, again there's this self verses nonself
how does this adaptive immune system distinguish host cells from foreign cells?
There's a tremendous potential for amplification, you can haveハ that we'll see my self proliferation,
and finally, and intriguingly there's this memory thing that you can have a first exposure
with some response, and then the re-exposure years later, decades later, can give you a
much stronger and faster response so you don't get sick to the sameハ in response to the
same pathogen the second time. So the main cellular players in the adaptive
immune system are a set of cells called lymphocytes. That arise from stem cells in the bone marrow.
Okay. And some of these cells, we will see, migrate
to the thymus, for further differentiation, and these becomes T cells. So T for thymus.
And in another set differentiate further within the bone marrow, these are called: B cells,
that's actually for fortuitous, the B was originally for the bursa, where these equivalent
cells in birds were seen but it works for us to call them bone cells.
And then, other of these lymphocytes third class go straight into the blood and become
natural killers NK cells that we've already talked about in innate immune system, what
did natural killer cells do? They respond to messed up host cells, right?
Okay. As opposed to foreign cells. So these are
the one set ofハcells, another set of cells are called antigen-presenting cells.
Around these areハ there's various cells that can serve this function in the adaptive
immune system including the dendritic cells and the macrophages that we saw in the innate
immune system and also these B cells can serve as antigen presenting cells and we'll find
out what this means shortly. So there's these basic questions, um, so adaptive
immunity, there's this, you know, specificity and diversity.
How is that achieved? How is memory achieved?
And how is the self verses nonself discrimination achieved in this system?
So, it apparently we're going to talk about the molecular bases of diversity first.
And the essential feature of this is that the B cells and the T cells, that are out
circulating in the lymph system, eventually, they inハ as part of their development from
the bone marrow cells they undergo gene rearrangements for specific genes on they're chromosomes
it's a one time process for each developing cell so all it's descendants will express
the same version of the rearranged genes and these are genes that encode for receptors
toハrecognize foreign molecules. Okay.
So these are receptors that are going to be on the surface of the B cells and T cells,
okay. I say it's a somatic gene rearrangement, because
this is one of the very few places other than gametogenesis where you have gene rearrangements.
So it's in the somatic tissues rather than in the germ line [Indiscernible] tissues.
So the receptors on B cells and the receptors on T cells are two different kinds of molecules.
Okay. They call them B cell receptors, T cell receptors,
they both arise by gene rearrangement but it's two separate kinds of genes, two different
receptor it is B cell receptors we're going to draw as Ys and the T cell receptors as
just straight bars. Okay.
So, the B cell receptors, looking at a little bit more fine grain molecular analysis, it
turns out that there areハ it's a tetramer, there are two heavy chains that are anchored
into the plasma membrane and two like chains. Okay.
And these floor protein chains are coupled to each other through disulfide bonds.
In each of these molecules there is aハ there's one region, near the membrane side, that's
called: The constant region or C region. So, pretty much every B cell would make the
same amino acid sequence for this sort ofハハ this part of the molecule.
Okay. And so both light and heavy chains have constant
regions. And then both light and heavy chains also have variable regions.
And it's these variable regions that are intriguing and it's the variable regions that generate
all different kinds of receptors for all different kinds of antigens.
Is there anybody in the back I'm about to lose my battery in this thing if anybody cares
about that. We'll make it through.
So, what happens is, let's look at these heavy chains the heavy chains are on chromosomeハ
the genes are in chromosome 14, so you've got two chromosome 14s, each of them has the
set of variable region, so there's little cassettes, repeats that are nonidentical,
there's a V, a D, and a J set of cassettes, and then there's also a conハsubstantiate
domain. So, what happens is, at a certain point in
the development of the immune system, thank you very much.
At a certain point in the development of the B cell, you have special enzymes that act
and they randomly cut out some of the Js and some of the Ds, okay, and connect the adjacent
parts together. Okay. So now you've got a D J region.
Okay. And all of the other versions of D and J are
eliminated. And then in a second step, you eliminate unwanted
V and D regions. Okay. And recombine and now you have a VDJ segment,
okay. And it called be anyone of the Vs with anyone of the Ds with anyone of the Js.
Okay. And this gives you a mix and match this is called: Combinatorial variation, okay.
So that just from looking at these simplified model you get at least 18,000 combinatorial
possibilities and that's essentially the number of genes we have in our whole body, so one
of to problems is, how can you make million different flavors of antibodies if we only
have 20,000 genes in our whole genome, right. And it's by this combinatorial process that
allows you to achieve that tremendous variability. Okay.
So you've got recombination to form, the heavy chain, you've got recombination to form the
light chain, there'sハactually two different versions of the light chains one on chromosome
2 and another set on chromosome 22. So you use or the other.
Okay. And there's an essential feature, now we've
got two sets of each chromosome, right? So, if you're having this recombination occurring
randomly, on each of those two chromosomes you would get two different flavors of heavy
chain, right? And two different flavors of light chain.
So, there's something else that's going on and that is a silencingハ so the successful
recombination on one chromosome, one chromosome 14, whatever, silences the other chromosome.
Okay. At this region.
So, recombination does notハoccur, the other chromosome never expresses its heavy chain
genes, so each normal B cell will only make one flavor of heavy chain and one flavor of
light chain. Okay.
Questions about this? That's an essentialハ
So, now, theseハ I should point out, the thing about the immune system we think about
antibodies. And the B cell receptors that's what we reallyハ
that's what an antibody is. The Y shaped molecule.
The T cell receptors have similarities but they're not antibodies. They're T cell antigen
receptors, not antibodies. Okay. So again you've got two different chains, alpha and
beta, linked by disulphide bridge, constant regions, variable region, here the alpha chains
are encoded on chromosome 14 the beta chains on chromosome 7 it doesn't matter that you
don't have to memorize that, you just need to know that these are different genes in
different positions in the genome, right? Whoa. I think that would be a good place to
stop. We're obviously going to go on with this on
Wednesday. So we're all set in terms of preparing for