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Biosci 201 > Final exam > Flashcards

Final exam Flashcards

1
Q

Signal hypothesis

A

secreted proteins contain a signal telling them where to go

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2
Q

How to test the signal hypothesis

A
  • Choose a culture a cell type which secretes proteins
  • fractionate to isolate ER
  • create a cell free system for in vitro translation
  • label proteins with pulse chase
  • purify an immunoglobulin and translate
  • disrupt membrane at different time points and examine size
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3
Q

Read out experiment

A
  • adding detergent to remove microsome
  • when microsomes were disrupted when proteins are still translated
  • longer base pairs found
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4
Q

Major protein sorting pathways in eukaryotes

A
  • cystolic proteins translated in cytosol but some will need to be targeted somewhere due to targeting sequence
  • proteins translated on ribosomes attached to RER but then translocated to lumen
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5
Q

Experimental evidence for signal hypothesis

A
  • direct relationship between a large precursor protein and small mature proteins
  • mature secretory protein only produced if microsomes were present
  • mature secretory proteins were protected from digestion inside microsomes
  • conserved sequence
  • found at N terminus which has 6-12 hydrophobic AA
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6
Q

Co-translational translocation process

A
  • signal recognition particles (SRP) binds to signal sequence to halt translation
  • SRP binds to SRP receptor on ER membrane via GTP hydrolysis
  • polypeptide transferred into Sec 61 translocon
  • polypeptide chain elongates and translocates through channel
  • signal sequence cleaved
  • polypeptide chain in lumen folds
  • ribosomes dissociates and channel closes
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7
Q

Sec 61 translocon

A
  • conserved protein
  • structure has a pore with a plug to form a channel
  • chain elongation at ribosome is sufficient to drive polypeptide through channel
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8
Q

Integral membrane protein path

A
  • ER
  • Golgi
  • Membrane
  • Lysosome
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9
Q

Where are integral proteins synthesised

A

RER

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10
Q

Topogenic

A

orientation and number of times a polypeptide crosses the membrane

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11
Q

Type 1 integral protein

A

COO- in cytosol and NH3 signal in ER

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12
Q

Type 2 integral protein

A

NH3 in cytosol and COO- in ER

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13
Q

Type 4 integral protein

A

have loops with NH3 in ER and COO- in cytosol

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14
Q

Getting proteins into the membrane

A
  • new polypeptide chain-ribosome complex associates with translocon
  • signal sequence cleaved and translocated
  • stop transfer anchor sequence of hydrophobic AA is translated and enters the translocon
  • no more translocation into lumen
  • stop transfer anchor moves laterally into membrane via translocon cleft
  • polypeptide anchor
  • translocon closes
  • ribosome dissociates
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15
Q

Mitochondria

A
  • contains own DNA but cannot make all their own proteins
  • grow and divide via the uptake of cellular proteins and lipids
  • pre proteins for matrix have amphipathic alpha helix signal sequence
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16
Q

Post translational translocation

A
  • pre protein synthesised on cytoplasmic ribosomes are kept unfolded
  • pre protein binds to Tom20/22 receptor on the mitochondrial outermembrane
  • Tom20/22 transfers pre protein to Tom40 pore
  • Tom40 passes pre protein to Tim complex in the inner membrane
  • pre protein transferred int matrix
  • Hsp70 chaperone binds and uses energy from electrochemical gradient to cleave signal sequence
  • protein now active in mitochondrial matrix
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17
Q

Methods to study secretory pathways

A
  • In vivo = radiolabel AA from secretory tissues
  • In vitro = live imaging fluorescent fusion proteins in cells
  • Conditional yeast mutants = force mutations in cell and look for phenotypes
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18
Q

Vesicle budding and fusion to form membrane carrying proteins

A
  • initiated by polymerisation of coat protein complexes
  • coat proteins bind to cytoplasmic tails of proteins sticking out of ER membrane
  • vesicles pulled out
    -cargo recruited to membrane proteins and gather in vesicles
  • vesicles uncoat in the cytosol exposing membrane proteins
  • vesicles move through the cytosol via motor proteins
  • vesicle fuse to the targeted membrane by SNARE binding
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19
Q

Formation of COPII vesicles from ER

A
  • Sar1 GTP protein binds to sec 12 receptor in the membrane
  • exchange of GDP for GTP to energise the protein
  • Sar1 undergoes a conformational change which makes the N terminus tail to stick into the membrane of ER
  • Sar1 binds to Sec23/24 (coat protein components)
  • vesicle formation
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20
Q

What causes uncoating

A

GTP hydrolysis which allows the vesicle to fuse with the golgi

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21
Q

What does having more coat proteins do

A

attracts more proteins with signal

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22
Q

How are vesicles moved along microtubule tracks

A

Motors

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23
Q

Anterograde motors

A

forward moving

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24
Q

Retrograde motors

A

backward moving

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25
Q

Movement from ER to golgi

A
  • Anterograde
  • CopII vesicle initiated by Sar1
  • Dynein motor
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26
Q

Movement from golgi to ER

A
  • retrograde
  • CopI vesicles initiated by Arf
  • Kinesin motor
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27
Q

CopI vesicle roles

A
  • initiated by Arf (GTP binding protein)
  • recycles back ER proteins
  • returns missorted resident ER proteins with retrieval signal
  • binds selectively to receptors based on pH
  • binds tighter under acidic conditions in the golgi and less then less acidic conditions (ER)
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28
Q

RER retention signal for soluble proteins

A

KDEL

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29
Q

ER export signal

A

Di-acidic sequence which is found in the cytoplasmic domain of membrane cargo proteins

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30
Q

ER retentional signal for membrane proteins

A

Lys-Lys

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31
Q

Signal that targets proteins to nucleus

A

Nuclear localisation

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32
Q

Cisternal maturation

A

when vesicles fuse with the cis golgi they form new cis golgi and move forward and everything gets pushed forward into trans golgi

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33
Q

Role of post translational modifications

A
  • quality control (tags misfolded)
  • structural stability
  • production of distinct molecules for signalling
  • activation/inactivation of enzymes
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34
Q

4 main post translational modifications

A
  • folding and assembly of multi subunit proteins in the ER
  • disulfide bond formation in the ER
  • glycosylation modification in ER and golgi
  • specific proteolytic cleavages to activate/inactivate occurs in ER, Golgi and vesicles
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35
Q

N-linked glycosylation

A
  • occurs in ER
  • precursor oligosaccharide (3 different branches of sugars) detects signal sequence and binds to protein
  • 3 sugars are removed by glycosidases
  • protein is now ready to move to the golgi
  • travelling to golgi then more sugars are added
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36
Q

glycosylation signal

A

Asn-X-Ser/Thr

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37
Q

Vesicles that bud from trans golgi structure

A
  • inner layer = adaptor proteins
  • outer layer = clathrin
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38
Q

adaptor proteins

A

bind cytosolic domains of membrane proteins to determine what cargo is to be transported

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39
Q

Dynamin role

A

polymerises around neck of bud and stretched neck untill it pinches off using GTP

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40
Q

Lysosomal sorting signal

A

mannose-6-phosphate

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41
Q

Lysosome

A
  • digestive and recycling compartments to break down waste macromolecules to monomer building blocks
  • has digestive enzymes which are active in acidic pH of lysosome
  • pH maintained by H+ pumps
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42
Q

Where is the lysosome signal added to the protein

A

cis golgi

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43
Q

Protein from golgi to lysosome

A
  • M6P receptor binds to M6P signal
  • protein recruited in clathrin/AP1 coated vesicles
  • receptors recycled
  • vesicle uncoated via Arf
  • change to acidic pH = endosome formaton
  • dephosphorylation and activation of protein
  • endosomes fuse into lysosome
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44
Q

Lysosomal storage disorders

A
  • Battens disease = mutation in gene coding for lysosomal enzymes which leads to accumulation of lipids as lysosomes cant form
  • No M6P signal
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45
Q

Regulated endocytosis

A

same process as lysosome transport but uses AP2

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46
Q

Regulated secretion of proteins from trans golgi

A

vesicles released due to a signal (insulin)

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47
Q

Continuous secretion of proteins from trans golgi

A

protein is always secreted (albumin), typically has no coat proteins

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48
Q

LDL signal sequence

A

Asn-Pro-X-Tyr

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49
Q

Receptor mediated endocytosis of LDL

A
  • LDL binds to LDL receptor
  • endocytosis
  • AP2 clathrin vesicle formation
  • targeted to endosome
  • dissociates from receptor due to pH
  • receptor recycled
  • lysosome formation
  • LDL broken down into AA, FA and cholesterol
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50
Q

Familial cholersterolemia

A
  • mutation in LDL receptor
  • increase in cholesterol
  • heart disease
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51
Q

LDL receptor

A

Type 1 transmembrane protein

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52
Q

Uniporter

A

movement of a single molecule down gradient

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53
Q

cotransporters

A
  • symporter and antiporter
  • couple transport of 2 different molecules
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54
Q

pumps

A

hydrolyse ATP to transport ions against their gradient

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55
Q

Similarities between channels and transporters

A
  • made of multiple membrane proteins that assemble in lipid bilayer to form an aqueous pore
  • regulated or gated
  • can undergo a conformational change
  • chemical energy coupled to movement
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56
Q

Peter agre

A

inserted mRNA aquaporin into frog egg and saw cell burst which indicated water movement

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57
Q

where are there a high concentration of aquaporins

A
  • kidneys
  • intestines
  • plant roots
  • in desert mammals kidneys to avoid dehydration
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58
Q

GLUT 1

A
  • a uniporter that takes glucose into the plasma membrane of cells through facilitated transport
  • has 2 conformational states which changes by glucose binding
  • limited
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59
Q

Vmax

A
  • maximum transport rate
  • achieved when concentration gradient is large
  • uniporter working at max rate
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60
Q

what determines the rate of transport

A

affinity

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61
Q

Km

A
  • affinity of a transporter for its substrate
  • concentration of substrate at which transport is half Vmax
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62
Q

GLUT 1 Km

A
  • decreased Km
  • efficient as high affinity for glucose
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63
Q

GLUT 2

A
  • uniporter in pancreas
  • high Km allows it to be a glucose sensor
  • low affinity for glucose
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64
Q

Why is there a low cytosolic concentration of glucose

A
  • rapid phosphorylation of glucose to G6P
  • allows constant import of glucose
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65
Q

GLUT 4

A
  • in muscle and adipose cells
  • stored in vesicles attached to golgi
  • insulin responsive
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66
Q

insulin and GLUT 4

A
  • insulin binds to receptors on muscle cells
  • signalling occurs
  • kinesin transports vesicles
  • GLUT 4 receptors are inserted into plasma membrane
  • increase glucose uptake into cell
  • decrease glucose concentration
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67
Q

What happens when there is low glucose

A
  • GLUT4 endocytosis
  • transport to endosome
  • GLUT4 recycling
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68
Q

sodium glucose symporter

A
  • transports glucose into cells when the outside concentration decreases
  • transports 2 Na down gradient and glucose against gradient
  • energy released by Na movement powers transport
  • found in intestinal and kidney tubule epithelial cells
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69
Q

Types of ion pumps

A
  • P class
  • V class
  • F class
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70
Q

P class pump

A
  • found in plasma membrane
  • tetramer
  • has catalytic and regulatory subunits
  • ionic composition of cytosol kept constant
  • Na/K pump and Ca pump
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71
Q

V class pump

A
  • found in lysosomes, endosomes and vacuoles
  • pump protons due to acidity differences
  • keep inside of lysosomes, endosomes and vacuoles acidic
  • balanced by facilitated diffusion of Cl to maintain electrical neutrality
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72
Q

F class pump

A
  • found in inner mitochondrial membrane
  • pump protons from matrix
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73
Q

Na/K ATPase

A
  • Na and ATP binds to pump
  • phosphorylation of alpha subunit
  • conformational change
  • 2K bind and 3Na out
  • dephosphorylation
  • conformational change
  • 2K into cell (cytosol)
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74
Q

Transcellular transport of glucose into blood

A
  • Na/K ATPase brings 2K in and 3Na out
  • K channel opens and K flows out to set up membrane potential
  • Glucose broken down from food in intestine and is moved into the cell by a glucose/Na symporter
  • GLUT 2 moves glucose from cell to blood
  • Aquaporin moves water from lumen to cell via osmosis
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75
Q

Acidification of stomach lumen by parietal cells

A
  • H/K ATPase (P-class) moves protons out of cell into stomach lumen, K into cell
  • K channel opens to allow K to flow back
  • Cl/HCO3 antiporter opens due to increased pH in cytosol, moves HCO3 into blood and Cl into cytosol
  • Cl channel opens and moves Cl from cytosol to stomach lumen
  • stomach lumen pH decreases as HCl forms
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76
Q

Electrical potential

A

differential distribution of charged ions on each side of the membrane

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77
Q

What interactions generate a membrane potential

A

K/Na ATPase and K channel

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78
Q

K channel structure that allows specificity

A

side chains of amino acids around the pore which only bind and interact with K

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79
Q

excitatory cells

A
  • neurones
  • muscle
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80
Q

Depolarisation

A
  • Na flows into cytosol down its gradients
  • Na channels open and voltage change triggers opening of gated Na channels down axon
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81
Q

Repolarisation

A
  • +50mV membrane potential
  • no more Na inflow
  • voltage gated K moved out of cytosol into exterior
  • Membrane potential now -70mV
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82
Q

what happens if action potentials arent regulated

A
  • toxicitiy in brain
  • seizures
  • over expression of inhibitory cells
  • parkinsons disease
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83
Q

Voltage gated Na channels

A
  • Has alpha helices with amino acids that are positively charged
  • when closed the alpha helices are attracted to negative of cytosol
  • alpha helices move towards negative exterior due to depolarisation which opens the channel
  • when +50mV membrane potential reached then channel inactivating segment blocks channel untill -70mV reached (Refractory period)
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84
Q

Optogenetics

A

uses light activated channels to control cell function through manipulating membrane potential

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85
Q

Channel rhodopsin 2

A
  • activated by blue light
  • Na moves into cytosol
  • stimulates AP
  • can control movement
  • used to study behaviours, parkinsons, epilepsy and PTSD
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86
Q

Synapse

A

point at which 2 neurons or cells meet

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87
Q

Neurotransmitters

A
  • chemical messengers
  • synthesised from amino acids in cytosol
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88
Q

Neurotransmitter release at synapse

A
  • neurotransmitters imported into vesicle using V class pumps and a proton antiporter
  • vesicles move towards membrane
  • V and T SNARES bind to form a SNARE complex to dock the vesicle at membrane
  • Action potential arrivs
  • voltage sensitive Ca channels open
  • vesicle uses with synaptic cleft via synaptotagmin binding with SNARE
  • neurotransmitter exocytosis into synaptic cleft
  • Na/neurotransmitter symporter reuptake of neurotransmitter
  • clathrin/AP2 recycles vesicles
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89
Q

Botulin toxin

A
  • prevents exocytosis of neurotransmitters at the neuromuscular junction as SNARES cannot fuse
  • 2 part polypeptide
  • binds to motor neurons to prevent Ach release = paralysis
  • protease which cleaves V-SNARE on vesicles so they cannot dock
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90
Q

cocaine/amphetamine

A
  • bind and inhibit DAT
  • increase dopamine in cleft
  • decreased reuptake of dopamine
  • increased mood
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91
Q

Antidepressants

A
  • act on serotonin reuptake symporter
  • increases serotonin in synaptic cleft
  • increased stimulation
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92
Q

Patterning

A

how cells develop their fate in space and time

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93
Q

Cell lineage

A

progressive determination of cells with restriction in developmental potential and differentiation into specialised cell types

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94
Q

How can cells of c.elegans be tracked during division

A

using non toxic dye which is shared to daughter cells

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95
Q

What type of cell division is c.elegans

A

asymmetrical

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96
Q

The orientation of what determines the nematode body

A

blastomeres

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97
Q

what determines the axis of cell division

A

mitotic spindld

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98
Q

What proteins are in the AB

A

Par 3 and Par 6

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99
Q

What proteins are in the P1

A

Par 1 and Par 2

100
Q

Par protein role

A

organising the distribution of other molecules and orientation of mitotic spindle

101
Q

Par3 role

A

inhibits centrosome rotation/rotational development in AB

102
Q

What determines cell-cell interactions

A

the positioning of cells

103
Q

Cell-cell interactions in c.elegans

A
  • P2 signals APX1 moleule to ABp which induces differentiation via notch signalling
  • P2 signals MOM2 to EMS via wnt signalling
  • ABa doesnt receive any signals
104
Q

Pop1 accumulation in c.elegans

A
  • Pop1 accumulates in mesoderm
  • pop 1 degraded in endoderm due to mom5
  • cell becomes polarised
105
Q

How to see if cell to cell signaling is important for development

A

seperate or remove cells to see the impact on differentiation

106
Q

Drosophila cell division

A
  • nuclear division without cytokinesis = karyokinesis
  • generates a syncytium (lots of nuclei within a cytoplasm)
  • cellularisation = nuclei migrate to egg surface and membrane forms around each
  • asymmetric distribution of mRNA at egg
107
Q

Determining factors in drosophila

A
  • anterior = bicoid which binds to hunchback and turns it on
  • posterior = nanos
108
Q

unique features of mammals

A
  • slow cell division
  • no maternal factors
  • rotational cleavage
  • asynchronus cleavage
  • cells end up in one of 2 microenvironments to determine fate
109
Q

Key events during early human development

A
  • fertilisation
  • cleavage
  • compaction
  • cavitation
  • implantation
110
Q

Morula

A
  • compaction of blastomeres
  • increased cell to cell adhesion
  • new cavity
111
Q

Compaction in mammals

A
  • cells flattening
  • increased cell to cell adhesion
  • formation of epithelium
  • formation of tight junctions between epithelial ells
  • becomes polarised
112
Q

when does differentiation start in mammals

A

32 cell stage

113
Q

inner cell mass

A

embryo fate

114
Q

external cells

A

fated to trophoblast

115
Q

cavitation in mammals

A
  • Na moves from uterine across apical surface of trophoblast
  • Na/K ATPase pumps Na from basolateral surface of tm- ophoblast into blastocyst cavity
  • increased osmotic pressure
  • Cl/HCO3 exchange between trophoblast and blastocyst cavity to maintain electrical neutrality
  • water moves into blastocyst
  • embryo swells
  • cavity forms
116
Q

blastocyst expansion

A
  • cavitation = embryo swelling
  • same number of cells and cell volume
  • different cell shape
  • increased blastocoel volume
117
Q

Epithelial expansion

A
  • radial cleavage via horizontal plane
  • increases number of cells
  • same cell volume
  • increases blastocoel volume
118
Q

Radial cleavage

A

generation of trophoblast cells with a polarised side

119
Q

Tangential cleavage

A
  • vertical plane
  • generation of inner mass cells
  • one non polarised
  • one polarised
120
Q

What happens if there are too few cells in the morula

A

all cells will be outside cells and no embryo develops

121
Q

Pluripotent

A

gives rise to embryonic tissue

122
Q

Totipotent

A

gives rise to whole embryo and placenta

123
Q

When do embryo cells become totipotent

A

8 cell stage (seen in mice)

124
Q

Implantation in embryos

A
  • trophoblast cells adhere to uterine epithelium
  • secrete proteins and hormones to the endometrium
125
Q

IVF

A
  • blastocysts are uterine transferred
  • embryo screening can be done for x linked diseases (cystic fibrosis + downsyndrome)
126
Q

Ectoderm fate

A
  • skin
  • nervous system
127
Q

mesoderm fate

A
  • muscle
  • skeleton
  • blood
  • kidney
128
Q

endoderm fate

A

gut

129
Q

how epithelial cells change shape

A

using an adhesion cytoskeletal belt which contracts

130
Q

epiboly

A
  • cells flatten and spread
  • thinner cell layer
  • increased surface area
  • increased cell substratum adhesion
131
Q

intercolation

A
  • cells intermix and spread to form a single sheet
  • change in cell to cell adhesions
132
Q

convergent extension

A
  • elongation of tissue mass
  • changes in cell to cell and cell to substratum adhesions
133
Q

invagination

A
  • localised areas of cell constriction causing buckling and bending of sheet
134
Q

involution

A
  • folding of cell layer after invagination
  • extensive
135
Q

migration

A
  • cells move away from edges of coherent mass (Single cells)
  • decreased cell to cell adhesion
136
Q

ingression

A
  • cells detach from epithelial layer and migrate into basal extracellular matrix
  • changes in adhesions
  • single cells drop out
137
Q

proliferation

A
  • cell layer expansion
  • can lead to localised growth at an edge or folding and bucking
138
Q

Frog grastulation

A
  • Cleavage
  • bottle cells invaginate
  • bottle cells movie via involution
  • ectoderm spreads via epiboly and intercolates
  • mesoderm stretches via convergent extension
  • new cavity forms
139
Q

Sea urchin

A
  • invagination of endoderm
  • convergent extension of endoderm
  • mesenchymal cells extend filopodium and tow archenteron via ingression
  • ectoderm cells pulled inside via involution
140
Q

spemann mangold organiser

A

region where bottle cells are found which signals to neighbouring cells

141
Q

difference between bottle cells and neighbouring cells

A
  • bottle = autonomous/ defined fate
  • neighbouring = condition/location dependent
142
Q

nieuwkoop centre

A
  • determines where gastrulation will start
  • when sperm enter egg it causes corticol rotation which moves maternal factors
  • movement of maternal actors activates wnt signalling
143
Q

Mesenchymal cell features

A
  • secrete ECM
  • flattened
  • motile
  • expresses vimentin
144
Q

epithelial cell features

A
  • polarised cell sheet
  • specialised tight junction
  • attached to basement membrane
  • expresses cytokeratin
145
Q

somite

A
  • form in anterior to posterior direction
  • ball of mesodermal tissue found near segmented vertebral column
  • staining with e cadherin you can see the new somites due to increase in epithelial cells wheras old one have increase in mesenchymal
146
Q

How to study EMT signals

A
  • genetic models
  • in vitro culture systems
147
Q

drosophila genetic model EMT

A
  • twist and snail genes are activated by maternal dorsal
  • twist turns on mesenchymal fate (transcriptional regulator)
  • snail turns off epithelial fate (transcriptional inhibitor)
148
Q

In vitro EMT system

A
  • grow epithelial cells and add growth factors to induce and EMT event
  • observation = down desmosomes, downn adherens junctions, down cytokeratins, up motility, up spreading, up vimentin
149
Q

where do MET events occur

A

uteric bud and metanephrogenic mesenchyme

150
Q

MET event in kidneys

A
  • metanephrogenic mesenchyme and uteric bud grow together
  • MM induces UB to branch
  • tip of each branch MM condenses to undergo a MET
  • epithelial cells differentiate, proliferate and elongate to start forming nephron
  • basement membrane degraded to form a constant tube
151
Q

stem cells

A

cells that divide to form one daughter that goes on to differentiate and one daughter retains stem cell properties

152
Q

Why we need stem cells

A
  • growth
  • renewal
  • repair
153
Q

Multipotent

A

gives rise to all cell types of a specific tissue

154
Q

unipotent

A

gives rise to one type of cell

155
Q

stem cells giving rise order

A
  • stem cell
  • restricted stem cell
  • prognitor cell
  • terminally differentiated cell
156
Q

Epidermis layers

A
  • basal layer = keratinocyte stem cells reside here to be protected, proliferation occurs
  • keratinocytes differentiate and move upwards
  • outer layer = keratinocytes flatten and lose their nucleus and die
157
Q

Drosophila stem cells neuroblasts

A
  • neuroblast (with bazooka, pins) drops out of epithelial cells via ingression undergo an EMT event
  • bazooka and pins = stem cell proteins
  • miranda accumulates
  • asymmetric cell division = one stem cell and one mother cell
158
Q

What can embryonic stem cells give rise to

A
  • epithelial cells
  • neurons
  • embryoid body
159
Q

transgenic mice using embryonic stems experiment

A
  • isolate single cells from blastocyst of black mouse and grow in culture
  • clone and grow single cell for many generations to generate embryonic stem cells
  • transfect embryonic stem cells with a transgene and antibiotic present marker
  • selects cells in presence of antibiotic
  • inject cells into blastocysts of white mice
  • transfer embryos into surrogate mother
  • pups will be a mix of black and white = chimera
  • chemira and white = grey mice (heterozygous)
  • grey and grey = black mice (homozygous)
160
Q

Transcription factors that keep embryonic stem cells in their pluripotent state

A
  • Oct 4
  • Nanog
  • Sox2
161
Q

what do the transcription factors do

A
  • activate genes for self renewal and pluripotency
  • repress genes that induce specific differentiation pathwars
162
Q

Medical applications with induced pluripotent stem cells

A
  • remove differentiated cell and grow in culture with yamanaka factors to induce stem cells
  • can be disease specific drugs or repair disease causing mutations
163
Q

problems with induced pluripotent stem cells

A
  • no consensus yet on most consistent or optimal protocol
  • check for culture induced changes aka activation of oncogenes
  • difference with methylated regions in IPS and ES so they have to be treated differently
164
Q

Components of plasma

A
  • blood protein, water and RBC
165
Q

Immunity

A

the ability to defend against infection and distinguish between self and altered self

166
Q

Antigen

A

a molecule that elicit an immune response

167
Q

Features of an immune system

A
  • specificity
  • diversity
  • memory
  • tolerance
168
Q

Types of immunity

A
  • mechanical and chemical defences
  • innate
  • adaptive
169
Q

Types of cytokines

A
  • chemokines
  • interleukins = differentiation and function of T + B cells
  • interferons = antiviral
  • tumour necrosis factors = cell death
170
Q

What can flow cytometry measure

A
  • fluorescently labelled antibodies
  • size
  • complexity
  • proteins secreted by a cell
171
Q

Flow cytometry method

A
  • cells are isolated
  • antibiotics added
  • stain for surface or intracellular markers
  • run sample
  • cells seperated by laser
  • graph plotted
172
Q

Primary lymphoid tissue

A

where cells are generated (T cells = thymus, B cells = bone marrow)

173
Q

secondary lymphoid tissue

A

site where adaptive immune responses are generated

174
Q

Stromal cells

A

provide structural support, organise discrete zones and secrete soluble mediators for cell survival

175
Q

Innate immunity

A

first line of defence = physical and chemical

176
Q

skin epidermis

A
  • produce a permeable barrier
  • multiple layers of keratinised epithelial cells
  • has a sebaceous gland to produce oils to prevent pathogens
  • sheds outer layers to remove pathogens
177
Q

antimicrobial peptides

A
  • defence peptides that are amphipathic with cysteine residues
  • secreted by phagocytes and epithelial cells
  • disrupts bacterial membrane by causing rupturing (Defensin)
178
Q

What is the complement system

A
  • 30 proteins travelling in the blood which are activated by infection
  • proteolytic cascade
179
Q

complement system pathways

A
  • classical = activated by antibodies
  • lectin = activated by lectin
  • alternative = activated by parts of pathogen
  • ALL ACTIVATE C3
180
Q

C3 activation results in

A
  • C3a and C5a chemoattractants activate and recruit immune cells
  • C3 binds to other complement proteins to form the MAC creating pores to kill cells
  • optimisation of pathogens = macrophage
181
Q

Different types of phagocytes

A
  • dendritic cell
  • macrophage
  • neutrophil
182
Q

dendritic cell

A

take up antigens and present on surface to lymphocytes

183
Q

macrophage

A

phagocytose bacteria and present antigens

184
Q

neutrophil

A

ingest pathogen and release enzymes to kill them

185
Q

what do phagocytes recognise

A

PAMPS using Pathogen recognition receptors

186
Q

Pathogen recognition receptors (PRR)

A
  • toll like receptors found at cell surface to recognise external components
  • can be inside for internal components
  • binds to PAMPS to allow for production of cytokines and pahgocytosis
187
Q

Natural killer cell activation

A
  • controlled by signalling between activating and inhibitory receptors
  • inhibitory = if normal cells are present, recognises MHC class 1
  • activating = if infected cells
188
Q

Signs of inflammation

A
  • redness and swelling due to vasodilation
  • pain
  • heat
189
Q

function of inflammation

A

eliminate cause of infection and create an environment for adaptive immune response

190
Q

inflammation response

A
  • bacterium expresses PAMPS which are recognised by dendritic cells
  • dendritic cells secrete cytokines and present antigens
  • natural killer cells kill infected cells
  • neutrophills lyse bacteria and release reactive oxygen species and phagocytose
  • antigen moves to lymph node for adaptive response
  • mast cells activated and secrete histamine which increased vascular permeability = swelling = vasodilation
191
Q

B cells

A

recognise intact antigens in lymph, blood plasma and interstitial fluid

192
Q

T cells

A

recognise antigenic fragments that are processed and presented by MHC molecules

193
Q

MHC class 1 and 2 similarities

A
  • encoded by HLA genes
  • bind peptides to present to the t cells
  • membrane proteins
  • peptide binding stabilises structure
  • lots of sequence variation = highly polymorphic
194
Q

MHC class 1 characteristics

A
  • single alpha polypeptide which spans membrane
  • has a beta microglobulin which doesnt span the membrane
  • epitope binds to alpha polypeptide
  • expressed in all nucleated cells
  • activates CTL/CD8 cells
  • endogenous antigens
  • peptides are 8-11AA long
  • coded for by 3 genes
195
Q

MHC class 2 characteristics

A
  • 2 identical polypeptide chains that both span the membrane
  • epitope binding side is both alpha and beta peptides
  • expressed by antigen presenting cells (dendritic, macrophages, B cells)
  • activates helper t cells/CD4
  • exogenous proteins
  • peptides are 13+AA long
  • encoded by 3 genes
196
Q

MHC restriction

A

complementarity between the peptide and MHC and also between the T cell receptor and MHC

197
Q

peptide loading for MHC class 1

A
  • dysfunctional proteins targeted for proteolysis with u6 addition signal
  • protein moves to proteosome
  • immunoproteosome cuts protein into specific lengths for MHC
  • peptides move into RER using TAP with ATP binding casette
  • peptides bind to MHC class 1
  • chaperones dissociate
  • MHC moves to cell membrane
198
Q

virus strategies to interfere with MHC class 1 immune detection

A
  • MHC not presented
  • unfunctional proteasome prevents protein being cut into peptides for MHC
  • adenovirus competes with TAP to prevent docking of MHC
199
Q

MHC class 2 antigen presentation

A
  • exogenous protein taken up
  • protein enters lysosome to be broken down
  • MHC class 2 assembled in ER
  • MHC class 2 transported to endosomes with invariant chain via golgi
  • invariant chain is cleaved off in endosome leaving a CLIP fragment
  • CLIP blocks binding of peptides to MHC
  • HLA-DM binds to MHC to release CLIP
  • peptides bind to MHC
  • MHC travels to cell surface
200
Q

cross presentation

A

where exogenous antigens can be loaded into MHC class 1 - occurs mostly in dendritic cells

201
Q

characteristics of adaptive immune system

A
  • specificity
  • memory
  • tolerance
202
Q

cellular immunity

A

cytotoxic t cells and helper t cells which are directed against intracellular pathogens

203
Q

humoral/antibody mediated immunity

A

b cells which are directed against extracellular pathogens

204
Q

clonal deletion

A

removal of self reactive lymphocytes

205
Q

clonal selection

A

a single lymphocyte gives rise to many lymphocytes that express the same antigen specific receptor

206
Q

clonal selection hypothesis

A
  • each lymphocyte expresses an antigen specific receptor
  • binding of antigen to receptor results in lymphocyte activation
  • activated lymphocyte will differentiate to produce effector cells with the same receptor
  • lymphocytes with self antigen receptor will undergo clonal deletion
207
Q

BCR vs TCR

A
  • BCR = light and heavy chain
  • TCR = alpha and beta chains
208
Q

t cell activation primary response

A
  • t cell enters lymph node via blood
  • t cell scans dendritic cell for cognate antigen on MHC
  • t cell is activated and induces proliferation and differentiation to effector and memory cells
209
Q

TCR complex

A
  • made of TCR, CD3 and CD4/8 coreceptors
  • bind to distinct MHC which amplifies signal from CD3
  • keeps proteins docked
  • induces signalling
210
Q

3 signals required from dendritic cell to activate CD4 T cell

A
  • signal 1 = signal from CD3 in TCR complex
  • signal 2 = co stimulatory molecules expressed by dendritic cells which promotes survival and proliferation
  • signal 3 = cytokines
211
Q

T cell secondary activation/memory response

A
  • binding of TCR to its cognate receptor-MHC complex is enough to induce response
  • allows CTL to respond to infected cells directly
  • CD4 activates B cells
212
Q

diminishing t cell activation

A

-upregulation of CTLA4 which inhibits function of T cell
- functions as immune checkpoint
- tumour cells upregulate PDL1 which binds to PD1 on T cell to prevent its action

213
Q

CTL/CD8+

A
  • kill infected cells expressing MHC1 loaded with their cognate antigen at the cell surface
  • done with high specificity
  • releases granzyme and perforin to induce death
214
Q

types of helper T cells

A
  • TH1 = drive cellular immunity and promotes macrophage function
  • TH2 = promote antibody production
  • TfH = makes antibodies
  • TH17 = involved in inflammation
  • Treg = inhibit immune responses
215
Q

discovery of antibodies

A
  • serum from guinea pigs injected with a sublethal dose of diphtheria toxin protects other guinea pigs that are exposed to a lethal dose
216
Q

passive immunity

A

immediate response that transfers antibodies from someone who has recovered from the disease, short term immunity

217
Q

active immunity

A

delayed and long term response when exposure to a disease results in an immune response that leads to antibody production

218
Q

Antibody isotypes

A
  • IgM = 1st antibody produced
  • IgA = in bloodstream and mucosal
  • IgE = for parasites and allergy
  • IgG = neutralises viruses
219
Q

polyclonal

A

response mediated by many B cells responding to different parts of one virus

220
Q

B cell primary response

A
  • activation requires more than 1 signal
  • dendritic cell acquires antigen and moves to lymph node
  • DC presents antigen to CD4/T helper
  • T helper differentiates and proliferates to be activated
  • T helper interacts with B cell to induce differentiation
  • Some B cells secrete IgM
  • Rest of B cells secrete other isotypes
221
Q

Germinal centres

A

specialised structure in the secondary follicle of the lymph node

222
Q

Germinal centres role

A

affinity maturation due to somatic hypermutation = selected B cells with highest affinity for cognate antigen

223
Q

Role of antibodies

A
  • neutralisation = prevents bacterial adherence
  • opsonisation = promotes phagocytosis
  • complement activation to increase phagocytosis and lyse bacteria
224
Q

ELISA indirect method

A
  • antigen attaches to the well via charge interactions
  • well blocked to prevent non specific binding to well
  • antibody added
  • secondary antibody added with conjugate enzyme
  • substrate added
225
Q

secondary immune response antibody isotype presence

A

IgG&raquo_space; IgM as it has a higher affinity due to affinity maturation

226
Q

Unimmunised cells

A
  • decreased frequency of B cells
  • IgM
  • low antibody affinity
  • low somatic hypermutation
227
Q

Immunised cells

A
  • high frequency of B cells
  • IgG and IgA
  • high antibody affinity
  • high somatic hypermutation
228
Q

Types of vaccines

A
  • Live attenuated vaccine (live pathogen which is less virulent)
  • Inactivated pathogen
  • protein subunit and VLPS (protein component of pathogen which lacks genetic material)
  • toxoid
229
Q

Adjuvants

A

stimulate immune response by activating innate receptors which increase co-stimulatory molecules

230
Q

Cancer characteristics

A
  • uncontrolled proliferation (autonomous growth signals and lack of growth inhibition)
  • escape from apoptosis
  • lack of senescence
  • angiogenesis (forms blood vessels)
  • metastasis and invasion
231
Q

How cancers arise

A
  • mutations
  • chromosomal rearrangements
  • chromosomal losses
  • epigentic changes
  • viral genes
232
Q

What causes changes in DNA

A
  • copying errors
  • chromosomal seperation errors
  • inheritance
  • viruses
  • mutagens
233
Q

Evidence for a single cell starting cancer

A
  • all tumours from a patient were DNA sequences
  • All cells detected a similar mutant genome from a single parent but some continued to mutate (clonal diversity)
234
Q

How proto-oncogenes can become oncogenic

A
  • overexpression of normal protein (gene duplication or overactive promoter)
  • point mutation
  • viruses have homologue copy which increases viral replication
235
Q

Signalling molecule oncogene = PDGF-B

A
  • normal platelets have a dimer of PDGF-A and PDGF-B bound to PDGF-R
  • V-sis has a homologue of PDGF-B that can still bind to the same sites
236
Q

Signal receptor oncogene = EGF-R

A
  • HER1 + HER2
  • signaling triggered by dimerisation of receptor when ligand is bound
  • HER1 oncogene = deletion of extracellular domain which brings membrane bound areas together so continuous signalling in absence of ligans
  • HER2 oncogene = mutation in transmembrane domain which brings receptors together = continuous signalling
237
Q

Intracellular transducers oncogene = RAS

A
  • normally is activated by GEF and inactivated by GAP
  • oncogene blocks action of GAP so it remains active to increase signalling and proliferation
238
Q

Ways to cause loss or mutation of tumour suppressor gene

A
  • mutation resulting in a frame shift
  • loss of chromosomes
  • loss of heterozygosity (missegregation or mitotic recombination)
239
Q

Retinoblastoma

A
  • familial or sporadic
  • involves loss of Rb = continuous signalling with E2F
  • familial group suggests inherited genetics
240
Q

Rb normal role

A
  • in early G1 Rb is bound to E2F
  • mid G1 Rb is phosphorylated
  • E2F released
  • Rb continues to be phosphorylated to prevent E2F binding
241
Q

p53 normal action

A
  • blocks cell division by inducing p21 which blocks CDK2
242
Q

p53 in cancer and mutations

A
  • cells will carry on dividing even in the presence of damaged DNA
  • HPV produces E6 which binds to p53 to ubiquitate it and degrade it
  • only one mutant p53 can disrupt its tetramer function = dominant negative
243
Q

metastatic disease can

A
  • interfere with normal physiology
  • take up too much space
  • starve you
244
Q

how to treat advanced cancer

A
  • surgery
  • chemotherapy
  • radiotherapy
  • immunotherapy
245
Q

cytotoxic t lymphocytes with cancer cells

A

can mistake cancer cells for virus infected cells due to the altered protein sequences so they will release granules with perforin and granzymes to kill them

246
Q

immune checkpoints

A
  • stops CTL to protect tissues from excessive killing
  • PD1 is common checkpoint
  • most immunotherapies block PD-1 pathway to increase the activity of CTL

Biosci 201 (2 decks)

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