1
Q
Somatic NS
A
always cholinergic – Acetyl Choline released from efferent motor neurone nerve terminals when reach muscle, binds to nicotinic acetyl choline receptor
- Sodium influx > depolarisation > AP
2
Q
Sympathetic NS
A
= 2 nerves
Preganglionic – Ach > Nicotinic Ach (binds to Ach on next neurone)
Postganglionic – Ganglia to organ (short) – releases noradrenaline – binds to adrenoreceptor
B1 = Heart / B2 = Lungs
M2 = Heart (Gai) relaxes – rest/digest
M3 = Bladder (Gaq) contraction
3
Q
Metabotropic receptors
A
- Agonist binds to serpentine receptor (spans 7x)
- G-proteins (GTPases) break GTP to GDP
- Heterometric = 3 subunits
4
Q
Gas
A
Stimulatory – CAMP dependent stimulus (production of CAMP by AC activation)
5
Q
Gai
A
Inhibitory – Inhibits AC
6
Q
Gaq
A
signals through activation of phospholipase C – activates protein kinase C
7
Q
Gao
A
Other – directly stimulates/inhibits ligand gated ion channels by activating K+ channels (hard to generate AP
8
Q
Micturition
A
Peeing is parasympathetic:
- Muscurinic Ach M3 = contracts bladder detrusor muscle/relaxes internal sphincter
Sympathetic= to prevent peeing:
- B2/B3 relax detrusor muscle/internal sphincter constricts
9
Q
Neuromuscular junction
A
- AP travels down motor neurone (depolarisation of membrane)
- Na+ channels open allowing Na+ influx = depolarise membrane – opens Ca+ channel
- Ca+ floods into cell leading to vescicle trafficking of Ach
- Ach fuses with plasma membrane – releases Ach into synaptic cleft
- Ach binds to Nicotinic Ach receptors
- Binding causes Na+ influx – localised change in membrane polarity (mini endplate potential) – depolarisation across muscle
- Opens voltage gated ion channels – Na+ influx starts muscle contraction
10
Q
Neuromuscular junction – reuptake mechanism
A
- AchE (acetyl choline esterase) breaks down Ach in cleft > choline + acetate
- Choline transported back up with co-transporter sodium where its repackaged into vesicles receiving an acetate from the acetyl co-enzyme A
- Cycle starts again
11
Q
Contraction
A
- End plate potential (AP) from NMJ travels down sarcolemma into T-tubule – depolarises membrane – triggers L-type channel opening = forces RyR open
- Calcium induced calcium release – it’s the Calcium that forces RyR open
- As it flows through – binds to Troponin C in sarcomere on thin filament = contraction
12
Q
Contraction steps
A
- ADP +Pi bound to myosin head
- Ca2+ binds to Troponin C
- Conformational change in thin filament switches filament on
- Myosin cross bridges form – myosin bind sites on actin
- Power stroke (pushes actin to centre of M line on sarcomere)
- Release ADP + Pi
- Myosin – ATPase binds ATP
- Cross-bridge (myosin head) detaches from actin
- ATP hydrolyses and myosin head cocks
13
Q
Ischaemic cascade: (stroke)
A
- Low O2 (due to thrombus) - reduced ATP production
- ROS generation + Ca+ release = unhappy mitochondria (produces lactate)
- ATP reliant pumps fail, and cell becomes depolarised allowing calcium to flood in
- Excess calcium = increased glutamate = increased calcium = oxidative stress
- Excess Ca+ generates free radicals, ROS and apoptotic cells = cell death
- Release of tissue factor from cell death = blood clotting
- Overall leads to much larger lesions in brain
- Cerebral oedema (excess accumulation of fluid) occurs due to the leakage of the blood brain barrier
14
Q
Reperfusion
A
- O2 returns, ATP SERCA starts
- ROS generated damages membranes (ER)
- Calcium overload of mitochondria and ER
- MPTP opens sodium – calcium exchanger NCX reverses
- Endothelial dysfunction
- Haemostasis/prothrombotic
- Pro-inflammatory cytokines released
15
Q
Clotting cascade
A
- Intrinsic pathway: Each factor in cascade catalyses the next into active form (12 catalyses 11-11a)
- Extrinsic pathway: 10 activated by activated 7 which is activated by 3 (Tissue factor)
- Fibrinogen in blood as cant clot just anywhere (changes to fibrin – acts as mesh catching platelets)
16
Q
Fibrinolysis
A
- tPa (serine protease) released in endothelium
- tPa activates plasminogen – makes plasmin
- Plasmin digests proteins creating clots
- During strokes = given recombinant proteins that act like tPa – break down clots
- Given anti-coagulants = inhibit coagulation (Herapin)
17
Q
Traditional cloning steps
A
- Vector
- Preparation
- Insert preparation
- Ligation
- Transformation
- Colony screening
18
Q
Non-pacemaker cardiac action potential
A
- Rapid depolarisation due to opening of fast Na+ channels (0)
- Initial repolarisation caused by opening of K+ channels (1)
- Inward Ca2+ movement through L-type channels (slow) which open when membrane reaches -40mV
- Plateau phase due to delayed repolarisation (2)
- Repolarisation – when K+ channels open and closure of Ca2+ channels open K+ channels (4)
19
Q
Pacemaker cardiac action potential
A
- Spontaneous depolarisation (pacemaker potential) caused by funny currents. T-type ca2+ opens. K+ channels close (4)
- Depolarisation of AP caused by increased Ca2+ conductance through L-type channels. T-type channels close (0)
- Repolarisation of the AP as K+ channels open and Ca2+ close. Hyperpolarisation reached. Na+ ions open, initiating phase 4 – also called funny currents or If (3)
20
Q
Penetrance
A
Probability that a person carrying a disease-associated genotype will develop the disease within a given time period
• Number of individuals displaying symptoms, divided by the number of individuals with a disease-causing mutation X 100
21
Q
Mechanism of antibiotics
A
- Inhibition of cell wall synthesis (most common mechanism)
- Inhibition of protein synthesis (translation – second largest class)
- Alteration of cell membranes
- Inhibition of nucleic acid synthesis
- Anti-metabolite activity
22
Q
JAK/STAT signalling
A
STAT = always present in cytoplasm waiting to be activated by JAK JAK = Activation stimulates – cell proliferation, cell differentiation, cell migration, apoptosis
JAK/STAT signalling: • Ligand binds (cytokine) • Receptor dimerization activates JAK phosphorylation of receptor • STAT binds to phosphorylated receptor • JAK phosphorylates STAT • STAT dimer forms • STAT dimer travels to nucleus • STAT dimer binds DNA and changes gene expression
23
Q
NF-KB pathway
A
- Ligand binds to the cell receptor on the cell surface membrane causing conformational change
- This phosphorylates iKK activating it
- iKK phosphorylates iKB
- iKBis tagged with ubiquitin and sent to the proteasome to be degraded
- iKB dissociates from NF-KB
- NF-KB translocates to the nucleus where it starts transcription
- When negative feedback occurs – iKBa is transcribed and inhibits NF-KB
24
Q
Phagocytosis Steps
A
- Phagocytes extend their membrane round the microbe
- Forms a phagocytic vacuole
- Microbe then exposed to lysosomal-independent killing mechanism
- The phagosome may also fuse with a lysosome, forming a phagolysosome
- The microbe is then exposed to lysosomal-dependent killing mechanisms
25
P53 normal cell pathway
* Mdm2 ubiquitinates p53
| * This sends it for degradation
26
P53 stressed cell pathway
* Mdm2 doesn’t ubiquitinate p53
* So, another kinase phosphorylates p53
* This leads to gene expression of p21
27
Recombinant protein production
* The CDNA from the gene of interest is inserted into an expression vector
* This is cloned into a suitable expression system
* The best construct/expression hosts are selected
28
Cell cycle stages
G0 = When a cell exists the cell cycle at the first restriction point
G1 = Metabolic changes prepare the cell for division. At a certain point (the restriction point) the cell is committed to division and moves into S phase
- Late G1 – first restriction point of cell
G2 = Metabolic changes assemble the cytoplasmic materials necessary for mitosis and cytokinesis
29
Steps of Wnt signalling (when presence of binding)
1. The Wnt ligand (secreted glycoprotein) binds to Frizzled
2. Frizzled becomes dishevelled
3. This mutates the destruction complex
4. No phosphorylation occurs and so the B catenin is stabilised
5. Transcription is permitted (of proliferation genes)
30
Steps of Wnt signalling (absence of binding)
1. No ligand binds to frizzles
2. APC, Axin and GSK-3B (The B catenin destruction complex) undergo phosphorylation
3. B-catenin acts as the transcription factor
4. This then undergoes ubiquitination
5. This then undergoes proteasomal degradation
6. Transcription is inhibited
31
Fat absorption
1. Lipid emulsified by bile
2. Lipases break down triglycerides into FAs and monoglycerides
3. FAs and MGs are packaged into micelles and absorbed by the microvilli
4. FAs and MGs are converted back to triglycerides
5. Triglycerides aggregate with cholesterol, proteins and phospholipids to form chylomicrons
6. Chylomicrons move into a lymph capillary, which transports them to the rest of the body
32
MAP kinase cascade Steps
1. Ligand binds to the receptor tyrosine kinase (RTK)
2. RTK dimerises and auto phosphorylates
3. GRB2 binds to the RTK
4. SOS binds to the GRB2
5. GRB2 binds to RAS (which has GDP bound to it)
6. SOS causes GDP to exchange to GTP, activating RAS
7. RAS binds to MAPKKK Ras and helps activate it
8. Raf phosphorylates and activates MAPKK Mek
9. Mek phosphorylates and activates MAPK Erk
10. Erk translocates to nucleus, activates transcription factors
33
RAAS Steps
1. BP decreases, so kidneys release renin into the blood
2. Renin converts angiotensinogen into angiotensin 1
3. Angiotensin 1 is converted to angiotensin 2 by ACE
4. Angiotensin 2 causes vasoconstriction, and the release of aldosterone by the adrenal glands
5. Aldosterone causes sodium retention and potassium excretion
34
Psoriasis development
1. A trigger e.g. pathogen causes Langerhans cells to become activated and phagocytose the trigger.
2. These migrate to the lymph nodes, secreting IL-1, IL-6 and TNF a
3. They present to a naive CD4 helper cell in the presence of:
a. MHC II
b. Co-stimulation with CD80/CD86
c. Cytokines IL-6 and IL-23
4. This undergoes clonal expansion and proliferates into Th17 effector cells
5. Th17 cells migrate to the dermis where they secrete IL-17 and activate keratinocytes
6. Keratinocytes secrete further IL-17 and the cycle becomes self-perpetuating
7. VEGF increases vascularisation and skin become psoriatic
35
IL-23 – Th17 – IL-17
IL-23 – Th17 – IL-17
36
IL-23/IL-17 inflammation Axis
IL-23 induces the differentiation of naïve CD4 cells into highly pathogenic helper T-cells (Th17) which produces IL-17, IL-6 and TNF a. IL-17 recruits’ neutrophils and pathogens to infected tissue.
- IL-23 promotes the proliferation and maintenance of the Th17 cells
37
Apoptosis
- The cells activate caspases that are normally dormant
- These caspases dismantle the cell from within
- The apoptotic cell breaks into blebs that can be engulfed by other cells
- This prevents the cell contents leaking out of the dying cell and allows the components to be recycled
38
IGF/Akt pathway
- Growth factor binds to tyrosin kinase molecule
- Receptor dimersiation and conformational change
- Transautophossphorylation
- Sticky patches attracts SH2 of PI3K phosphorylates PIP2 to PIP3
- PIP3 recruits protiens with particular binding sites - PH
- PIP3 recruits PDK1
- Phosphorylates Akt
- Akt binds to PIP3
- Akt further phosphorylates mTOR
- Akt now super active, phosphorylates others
- Kinase cascade
39
GABAa channels
Causes hyperpolarisation which reduces excitability
| - Ligand gated chloride channels
40
GABAb channels
Inhibits VOCCs and opens K+ channels = reduces excitability
| - Gi/Go
41
Withdrawal reflex
Pain signal travels along the Ad fibre, to the dorsal root ganglion
- Travels across an interneuron to the anterior horn cell
- Travels along a motor neuron to move e.g. the hand
42
Peripheral sensitisation
- Sensory neuron expression of a-adrenoreceptors, so more stress means more pain, and over expression of V-G Na+, which increased AP production and increases stimuli above the original injury
43
Clearance calculation
([Drug] in urine X urine flow rate / ([Drug] in plasma)
44
Cheng-prusoff equation
Used for: The IC50 value is converted to an absolute inhibition constant Ki using the Cheng-Prusoff equation
45
Steps of autophagy
1. Cytosolic material is sequestered by an expanding membrane sac, the phagophore,
2. Formation of a double-membrane vesicle, an autophagosome
3. The outer membrane of the autophagosome subsequently fuses with a lysosome,
4. Inner single membrane of the autophagosome exposed to lysosomal hydrolases
5. The cargo-containing membrane compartment is then lysed
6. Contents are degraded
46
Apoptosis- intrinsic pathways
1. Cytotoxic stress
2. Translocation of pro-apoptotic Bcl-2 family to mitochondria
3. Mitochondrial cytochrome C released
4. Oligomerisation of pro-apoptotic factor Apaf-1, forms apoptosome
5. Recruits caspase 9, which activates caspase 3
47
Apoptosis- extrinsic pathways
1. Fas ligand binds to death receptors
2. Protein recruitment and from death-inducing signalling complex (DISC)
3. Activates caspase-8 initiator
4. Activates caspase 3/7 executioner
what is the equation for radioligand equilibrium
kon = koff
What are MAO inhibitors
a class of drug which inhibit the activity of one, or both monoamine oxidases; both MAOs deaminate the neurotransmitter dopamine
48
The nigrostriatal pathway
key dopamine pathway
cell body in the substantia nigra, moves up to corpus striatum
Involved in the production of movement, as part of the basal ganglia motor loop
49
The tuberohypophyseal pathway
Dopamine release in this pathway is to inhibit prolactin release
50
Deep brain stimulation
A neurostimulator is implanted into the patient’s brain- subthalamic nucleus or Globus pallidus interna.
When switched on, the internal electrodes deliver high frequency stimulation to the targeted area, changing electrical signals which cause symptoms of diseases such as Parkinson’s.
51
Haemagglutination inhibition assay (steps)
1. Virus binds to RBCs
2. Specific antibodies prevent shield forming
3. Virus cannot cross link with RBC when antibody bound
4. RBC fall to the bottom of the well- button
52
Haemagglutination assay (steps)
1. Virus binds to RBCs
2. Cells fall to the bottom of the wells due to gravity
3. Sialic acids bound to by virus, forming a shield at the well bottom
4. Perform serial dilution
5. The last well with a shield means you can calculate the virus titre
53
Viral replication (stages)
1. virus adheres to cell surface
2. virus enters cell by pinocytosis
3. virus sheds coat
4. replication of viral nucleic acids
5. synthesis of viral protein of capsid
6. assembly of new virions
7. release of virus
54
Baltimore classification system
How viruses nucleic acids make mRNA
- There are 7 groups in the Baltimore classification system
- Flu are classification 5
55
Direct immunohistochemistry
1. Buy antibody specific for protein of interest
2. Antibody labelled/conjugated with ‘reporter’ normally florescent
Advantages:
• More specific – only binding to one antigen
• Involves fewer steps
• Useful when antigen is abundant
Disadvantages:
• Expensive – requires access to different specific Abs
• Label may interfere with Ab binding to epitope
56
Indirect immunohistochemistry
1. Buy antibody specific for protein of interest
2. Primary antibody binds to antigen
3. Secondary antibody raised against own lg from different species and labelled
```
Advantages:
• More sensitive
• Signal amplification
• Cheaper
• Useful when antigen isn’t abundant
```
57
Immunostaining
Process:
1. Block
2. Wash with PBS-Tween
3. Incubate 1*
4. Wash with PBS-Tween
5. Incubate 2*
6. Wash with PBS-Tween
7. Visualise signal (conjugate enzyme) via colour or fluorophore
Block: Prevents non-specific proteins from binding
58
Western Blotting
What is it: Use of a specific antibody to detect and quantify a particular protein in a sample
Sample type required:
- Mix of proteins in a tube
Steps:
1. Take cells from sample
2. Homogenise cells (mash them up) or Lyse cells
3. Run SDS (detergent) Page gel separate proteins by size, not charge
4. Transfer/blot cells
5. Wash, Add primary antibody
6. Wash, Add secondary antibody with stain*
7. Visualise results
* can do direct/indirect like immunohistochemistry
59
PCR
Steps of PCR:
1. Add buffer and Double stranded DNA template
2. Add dNTPs
3. Design primer- short nucleotide primer complementary to your gene of interest
4. Run PCR ~30 times
5. Denaturing stage 95*C
6. Annealing stage 55*C
7. Add specific primers
8. Extending stage 72*C
9. Add Taq polymerase (heat stable)
10. Run PCR on agarose gel electrophoresis
11. Visualise e.g. SYBR-Green
Steps of RT-PCR:
1. Isolate RNA from sample
2. Convert to DNA using reverse transcriptase
3. Denature at 98*C
4. Anneal at 55*C
5. Add specific primers
6. Extend at 72*C
7. Taq polymerase extends primers adding nucleotides
8. Cycle repeats to amplify DNA
9. Visualise the signal
RT-PCR vs. PCR:
- RT-PCR requires Reverse transcriptase to convert RNA to DNA
60
ELISA
Purpose: Uses specific antibodies/antigens to detect and quantify a particular protein or molecule
Steps:
1. Wells are coated in antigen of interest
2. Wells filled with dilutions of the patient’s serum
3. If antibodies against the antigen are present, they will bind to the antigen fixed to the bottom of the wells
4. Only antigen-specific antibodies will bind to the wells
5. Wells are washed out to remove all unbound antibodies
6. Animal antibodies against the human antibodies is added
7. These antibodies are covalently conjugated to an enzyme
8. The wells are then washed again, to remove any unbound enzyme-conjugated antibody
9. Solution of colorogenic enzyme substrate is added
10. The interaction of the substrate with the enzyme on the second antibody generates visible colour
11. Development of colour in the wells can be seen with the naked eye, or electronic plate reader
61
Flow Cytometry
What is it: A technology used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser
Measures: Fluorescence intensity from fluorescent-labelled antibodies, against specific proteins or antigens
```
Procedures its used to perform:
• Cell counting
• Cell sorting
• Detection of biomarkers
• Protein engineering
```
Steps:
1. Sample added
2. Hydrodynamic focusing as cells pass through in single file
3. Fluorescence emitted from stained cells
4. Scattered light by cells detected
DDT pathways
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