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N
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Looking at the kinetics behind drug transport through epithelial cells as well as drug
interactions with receptors to produce an immune response
...

Drug action – an introduction
Drug classification
-

Chemotherapeutic drugs which include treating infectious diseases and cancer

-

Pharmacodynamic drugs which treat non-infectious diseases (such as sedatives)
Miscellaneous is a subdivision of pharmacodynamic drugs and includes examples
such as analgesics and anaesthetics

Drugs names are either chemical, brand, trade, generic or common
...

- Some drugs can also be used to diagnose conditions
Disease classifications:
-

Infections – typically bacterial or viral
Allergic – immune responses
Metabolic – any process controlled by enzymes or receptors, typically refers to
biological pathways
Cancer – mutations within DNA
Toxic – poisons
Psychosomatic and mental conditions – usually related to environmental, social and
economic factors as well as neurotransmitters (such as dopamine and serotonin)

Sites of drug action
It is possible to get drug receptor interactions – receptors are typically found on cell
membranes, although there is a type of receptor which does not form on the cell membrane
and is found within the cytosol of the cell
...

Other sites of drug action are enzymes which can be membrane bound or free
...

-

These interactions can be reversible which means the enzyme can regain its
activating
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-

A drug can be competitive meaning it competes to bind with the enzyme in place of
the substrate
...
This enzyme breaks down neurotransmitters,
therefore Prozac improves these levels
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Non-specific interaction:
-

Non-specific drug interactions typically occur outside of cells
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Use a basic drug to neutralise the acid within the stomach
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The drug must be designed
to be more toxic to the organism that to the host
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Drugs can
be agonists (biological response seen) and antagonists (no response seen)

Pharmacodynamics – Branch of pharmacology that deals with the all the biochemical and
physiological effects of the drugs and their mechanisms of action
...

Introduce a ligand into the body which is designed to interact with this receptor
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This should be strong enough to warrant binding,
but not too strong as that the ligand cannot dissociate
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This ligand binding can induce a conformational change, which could activate an effector
system (a biochemical pathway)
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Once the effector system is activated it will go on to produce an effect
...
The biological
response can also be measured via efficacy
...
The amount bound to the
receptor and not bound can be measured
...
dm-3 – this is receptor affinity
Lfree + Rfree = LR

Drugs acting full/partial agonists/antagonists

-

-

Methadone is a full agonist shows a steady increase in biological response until a
maximum is reached
...

Partial agonist is still producing a response, but the maximal response is diminished
compare to the full agonist
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Problem with the saturation curves is that similar responses are seen to begin with
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Antagonists produce no biological responses
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Binding data
-

The rate at which a drug (L) combines with receptor (R) is dependent on the
concentration of both the drug and receptor
...


-

Ligand wants to bind to R, but may also bind to non-specific receptors too (such as Ra
and Rb)

The difference between the two types of binding is represented by line C on the
graph
...


Spare receptors
-

When a ligand is introduced, and it interacts with a receptor or enzyme, the
biological response will gradual increase into a maximal response is reached
...

For example, only 10% of acetylcholine receptors need to be activated to see a
maximal response and produce muscle contraction
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This induces a quick response and is biologically efficient
...


-

Within the cell is cytosol, which is encapsulated by the cell membrane
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-

Integral proteins can be receptors, these receptors can undergo chemical changes
and produce a chemical response
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Different receptors have a different number of
passes, some can come together to form a pore
...
Therefore, it is
important to understand how they are embedded, but the membrane must be
disrupted to see this
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Type 4 are nuclear receptors, these are
not membrane bound
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Of type 1, 5 of the
subunits come together to form a pore
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Also has an additional protein on the intracellular side
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Enzymes a typically involved with the regulation of
phosphorylation
...
This is key in
regulating biochemical processes
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-

Type 4 are receptors located within the cytosol
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Type 1
4 passes over the cell membrane with 5 subunits
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2 ligands must be
present to activate the channel
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This produces rapid responses
...
g
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The ligands are agonists
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Receptor
Embryonic – alpha1, beta1,
sigma and 
Adult – alpha1, beta, sigma
and gamma
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There is an additional protein on the intracellular side which is the Gprotein
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This is slower
than type 1
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Type 2 ligand will come
in and will induce the G-protein to dissociate from the receptor (if an agonist)
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The alpha subunit will dissociate and is now active from the beta and gamma
subunits, which remain bound
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These then control/regulate other biochemical pathways
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Type depends on the region of the body
where they are located
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Overall action is always a change in phosphorylation
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-

Type 1 gives millisecond responses, there are typically involuntary actions and
therefore an instant response
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Usually voluntary controls
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g
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Type 3
Receptor = insulin receptor and insulin growth factor R
Endogenous ligand = Insulin (IGF1, IGF2)
Response = Glycogen, triglyceride and protein accumulation and reduced glucose
-

Insulin response is enzyme link and relates to phosphorylation
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There are 2 single receptors that once phosphorylated induce a
conformational change and the 2 protein bound sections dimerise together
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An example is a
hormone such as oestrogen which must cross the cell membrane
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This produces a complex which can cross the nuclear membrane to enter
the nucleus and interact with DNA
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Then being able to bind to a
specific piece of DNA, either the gene on or off, which leads to protein production
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It protects us from foreign bodies such
as bacteria and viruses
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The
immune system consists of white blood cells, the lymph nodes, antigens and antibodies
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There is also a defence mechanism within the nostrils, through hairs that are
covered in mucus which act as a defence system against minute particles
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The tonsils are made of immune cells which protect against airborne pathogens as
well as food pathogens
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When the immune system is activated through B cells, T cells and antibodies as well as
bacteria pathways, the symptoms associated with illness can be seen
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Immune system
can eat cells to eliminate them
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The immune system must differentiate between self and non-self-cells
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Different cells have different tasks, they teach other cells within
the immune system how to recognise them
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Antigens are the foreign cells (non-self-cells) – they are proteins on the surface of
(pathogens) bacteria, fungi and viruses
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Pathogens and defence cells have surface proteins, when these
interact they give an immune response
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Allow for small particles
to be caught and digested
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Cancer drugs can be injected within the lymphatic system and transported around
the body
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Lymph nodes filter the lymph system to removed pathogens and waste products
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This is why they become enlarged
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Spleen – acts as a filtering system
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Skin – also secretes oils as an additional barrier and skin has a collection of bacteria
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There are different types of bone
marrow tissue that produce different types of cells
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They are released and mature into B cells and
T cells
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Everything we eat
will go through the GI tract
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Thymus – Helps to regulate the production of various immune cells
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Types of cells associated with the immune system
Adaptive defence system
3 primary defence cells: B cells, T cells and antibodies
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Innate defence system
Include mucous membranes, skin and scavenger cells
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Defence cells
-

White blood cells are a type of immune cells also referred to as leukocytes
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1
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Lymphocytes: produced within the bone marrow (red), can develop into B
cells, they can also move into the thymus and develop into T cells
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Different phagocyte cells have different breakdown
mechanism
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The pathogens are encapsulated within the phagocyte cell
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Oxygen dependant intracellular mechanisms
Two key types of killing mechanism – superoxide and myeloperoxidase (both utilise
enzymes within the pathway)
Super oxide (this is naturally produced) More efficient
- Super oxide = O2- is a by-product of oxygen metabolism
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- Superoxide can also react with hydrogen peroxide to produce a hydroxyl radical
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Therefore, they act as antibacterial
reagents, by disrupting the bacterial processes, killing them
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- Key enzyme in the production of hypochlorous acid HOCl – which acts as an
antimicrobial agent
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Causes oxidative
damage to host tissue
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Which is
where the source of oxygen comes from
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Oxygen independent intracellular mechanisms (typically utilise enzymes)
Not as efficient as dependant intracellular mechanisms, but processes do not require a
direct input of O2
Four main types using:
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Electrically charged proteins – can interact with bacteria cell wall to change the
charge
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Proteases – cleave amino acids breaking down protein structure
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Many receptors and enzymes are made of
proteins
...
Extracellular mechanisms

(pathway that can be activated by an immune response, to produce highly reactive nitrogen
species that can interact with cells that are nearby e
...
pathogens)
Nitric oxide – an extracellular pathway
- Produced a highly reactive metabolite ONOO- (this molecule causes DNA damage)
- Pathway is very similar to oxygen type pathway as both produce reactive species
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All of which take place within the phagocyte allowing for the death of a
pathogen
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Nitric oxide helps to regulate blood pressure, this takes place
out of phagocyte cell
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Reactive species is antimicrobial
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Interact with bacterial DNA and disrupt
reproduction
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(Nitric oxide synthase – typical enzyme found in
the nitric oxide pathway, superoxide dismutase, myeloperoxidase)
4
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The immune cell picks up an antibody (a memory of said pathogen)
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Allows for a secondary response, which is much quicker
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Good at taking up
dead/dying cells
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Responsible for releasing histamine
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Have surface proteins, call IGE (essentially an antibody)
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Once activated they can release basophil granules and histamine which can act on
histamine receptors which can be used to activate biochemical pathways
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This then attaches to the surface of the mast cell, making is susceptible to attack
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They help to promote the production of more antibodies
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-

Also produce lipid mediators, leukotrienes and prostaglandins
...
g
...
At the ends they have specific
protein structures and sequences to a particular antigen
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- They are secreted by B cells and can be found free or bound to the B cells
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B cells and T cells
Key functions:
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B cells: type of white blood cell, mainly come from red bone marrow (where they
mature) and main job is to secrete antibodies
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Tc cells are killer T cells that destroy virus infected cells
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They
mature within the thymus
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A main
function of histamine as a neurotransmitter is to help activate and regulate
acetylcholine
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-

Histamine is derived from the histidine amino acid but without the carboxylic acid
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Histamine:

-

Histamine has amine functional groups that can be targeted by other enzymes
within the body
...
Excess
levels of histamine are broken down by MAO-B
...
g
...


-

In order to get histamine release, antigens are needed to bind to antibodies which
are attached to mast cells
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Nmethylhistamine can also be converted to imidazole acetaldehyde (Using DAO
enzyme)
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So metabolised products can block the activity
of the key enzymes, leading to an increase in histamine, which cannot be broken
down
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Monoamine oxidase (MAO)
Neurotransmitter example:

-

Have amine functional groups that convert to acid functional groups
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Type B is associated with histamine breakdown
and is located within the mitochondrial membrane
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-

MAO can also break down dietary amines such as tyramine which is found within
foods
...
Excess of tyramine leads to high blood
pressure, so breakdown is needed
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If inhibited when have a histamine intolerance could lead to
issues
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1
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H2 – causes smooth muscle relaxation and can stimulate gastric acid
production
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H3 – affect acetylcholine levels (decrease)
4
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(chemotaxis
is the movement of a cell)

-

H1-H4 receptors are a class of G-protein coupled receptors
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-

When histamine interacts with receptors it can lead to different biochemical
pathways
...


-

Histamine receptors are very similar to dopamine receptors which are also G-protein
coupled receptors
...
On the intracellular side there will be a G-protein
...

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Doxepin is an antidepressant with antihistamine properties
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It is used as a reference standard in the quantification of
histamine and receptor interactions
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Shows examples of neurotransmitters binding to proteins
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(All activated via the alpha subunit)
-

Gs biochemical pathway increases phosphorylation
Gq pathway increases protein phosphorylation in addition to calcium release
Gi pathway decreases phosphorylation

All help to regulate the body’s immune response
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Allows the g-protein to dislocate from the
receptor
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-

Histamine then interacts with histamine receptor (e
...
H1), leading to a biochemical
response i
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vasodilation and changes in permeability

-

Histamine binds to the H1 receptor and promotes conformational change on a G
protein, which allows the alpha subunit to dislocate and produce active GTP
...
IP3 which has 3 phosphate molecules associated with it gets
broken down to open up the calcium channel
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DAG activates protein kinase C, which is also activated and regulated by calcium
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Overall purpose is to help regulate gene activity
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They essentially block the
ability of histamines to interact with the receptors as they have a higher binding
affinity to the receptors than histamines
...
(They preferentially bind to H1 receptors)

Other effects of antihistamines:
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Possible to use anti-histamines to treat cold symptoms
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These are involved in the transmission of
signals associated with pain
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Histamine roles
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Effects vasodilation and blood pressure
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(This is a good way to eliminate waste products)

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Histamine is also involved in the sleep/wake regulation
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If when issued an anti-histamine, an interaction with a H1 receptor increases
drowsiness and H3 increases wakefulness
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Histamine helps to regulate acetylcholine, which is a neurotransmitter
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pH within the stomach is around 2
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Negative feedback then stops the histamine release when the stomach
is at ideal acidity
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Histamine also protects neurons within the brain
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There is a link between histamine and schizophrenia
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This may be
linked to the neurone protectiveness of histamine
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Histamine increases permeability so high amounts
causes high permeability
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This is then allowing the passage of cells and bacteria
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MS sufferers have high levels of
histamine
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Conditions related to histamine release and deficiency
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There are over 23 functions of histamine including: vasodilation, effects on mucous
membranes and gastric release
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Role of histamine in allergic reactions
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When an antigen (or allergen) is detected and absorbed by our body, it prompts our
beta cell to produce antibodies
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Histamine then increases permeability (leakage of fluid) and causes swelling of blood
capillaries and a surface enlargement
...

Excess of immune cells are leaked into the area, increasing water volume and leakage of
other cell contents, dependant on permeability of the cell
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Excess histamine metabolites can inhibit HNMT
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Can get nausea, headaches and skin flushing
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This is due to high levels of histamine within red wine, a result of a bacterial
fermentation process
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This may be linked to histamine intolerance – a deficiency in the enzyme DAO
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Then histamine measures will
be measured before consumption, during consumption and then ether 12/24 hours
after
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Biological responses are due to excessive immune cells
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Causes high temperature and aching body
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High release of cytokines causes a cytokine
storm
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Antibodies produced in response to virus
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Results in chemotaxis – mast cells accumulate at site of infection
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This causes a build-up of fluid within the lungs
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When these particles break down the
pathogens this causes a green colour when coughing up mucus
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Quantification methods
All of these assays are designed to analyse how a drug can affect the opening and closing of
ion channels
...
Assays being
looked at are looking at quantifying changes in calcium levels
...

- These are difficult to manage and expensive, they also give a lot of background noise
radiation
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Radio-ligand assays are closes linked to scintillation assays, allows for the detection of radioactive decay from isotopes
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This would be a good way to monitor if a receptor had been activated or not

What is fluorescence?
Absorption of electromagnetic radiation will result in an electron being excited (either
electronically or vibrationally) to a higher state
...

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If light is absorbed around 400nm, the electron that drops will emit energy at around
600nm

-

A light source is used for fluorescence-based assays
Sometimes emission can be seen in the visible region of the electromagnetic
spectrum (Resulting in colour changes)
Fluorescence tends to follow 1st order kinetics
Jablonski diagram

-

Fluorescence is also linked to luminescence

Fluorescence spectroscopy
-

Uses a light source to emit energy

-

This light is polarised to a single plane when passed through a monochromator

-

Sample absorbs energy allowing excitation of an electron and then emits energy
when drops back down

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Detector analyses the wavelength which has been emitted

-

HTS (high throughput system), shows that multiple samples can be analysed at once

-

If a molecule within the sample is fluorescently tagged it will interact with the
polarised light disrupting its vibrational plane
...

This gives a higher chance that the polarised light will remain polarised
...
Interaction of this with a ligand causes a
conformational change and allows the alpha subunit to dissociate
...

Calcium binding assay then utilises the increase in calcium
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Which then binds to intracellular
calcium
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(490nm-514nm)
- More calcium should lead to a gradual increase in fluorescence
...
The atmospheric conditions and nutrients must be controlled
...
The fluoforte- is then able to enter the cells and becomes subjected to the cells
esterase
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This then leads to an increase
in fluorescence
...
This should be determined prior
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- As calcium concentration increases as does fluorescence intensity
-

Peptide displacement: used to screen compounds

Based on the same theory as above, still requires the polarisation of a light source to give
polarised light of a single plane
...

Green represents a peptide which is fluorescently tagged
...

Investigating if the peptide can be displaced by the addition of a compound
...
If different concentrations of compound are added, displacement of the
peptide can be analysed
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- Typically use fluoroscene

-

FRET: measures changes in proximity such as protease activity

Good for monitoring distance interactions
...
The ligand is excited at 400nm to emit a wavelength of 500nm, if bound to the
receptor the ligand is still excited at 400nm, but transfers energy to the receptor
fluorophore
...

Allows for the identification of receptor binding sites
...
There is also an issue with adding a fluorescent probe, which could alter the binding
affinity for the RL complex
...
A protease assay is prominent in detecting the
presence of arthritis in an individual
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This causes inflammation and arthritis
...
Proteins are made up of amino acids which
can be cleaved by protease
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One molecule is excited which then transfers
energy to the acceptor molecule resulting in an emission at a longer wavelength
...

-

Fluorescence correlation spectroscopy: analysis of fluctuation of the fluorescence
intensity

This is a good example for very small volume assays (1-2 molecules)
...
A relatively cheap detection method

The small volume well contains a couple of molecules which can diffuse freely throughout
the volume
...
If
the fluorescently tagged compound comes into contact with a receptor it will bind reducing
movement, slowing the signals produced
...
When ligand (agonist) interacts with
the receptor the alpha subunit dissociates and produces biological effects
...
This results to the recruitment of the compound beta-arrestin
...
Preventing reactivation and the production of more biological effects
...
beta-arrestin molecule has a protease function,
allowing it to cleave a protein sequence
...
(transcription factors regulate DNA
activity)
...
(which is added)
In the absence of beta-lactamase the coumarin molecule is excited at 400nm, this energy is
then transferred to the fluorescein (the fluorescent probe) which emits at a wavelength of
545nm
...

- Therefore, would see the coumarin only excitation of 460nm
...
When fluorescein and coumarin are bound the colour will be green,
whereas when cleavage occurs a blue colour will be seen
...

Fluorescence vs radiolabel
-

-

Fluorescence based assays are less hazardous and produce less waste
Can use a high throughput system and is much quicker
Can avoid a separation step, whereas in radio-labelled assays the bound and free
must be separated
Fluorescence targeting can alter the binding affinity of a ligand (K D)
Fluorescence is also sensitive to solvents- so must determine the effect of the
environment where the cells are incubated
...
This is also
linked to FRET analysis
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Investigates the binding of antibodies with anti-antibodies
...

Immuno assay is based on antibodies binding to each other
...
When this antibody is excited with a low wavelength it can emit
energy at a higher wavelength but can also transfer some of its energy to another antibody
via binding
...

If an agonist activates a type 2 receptor this can affect cyclic AMP levels
...
Assuming an increase in cAMP
which is present as a free molecule within the cells
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It is known that this anti-antibody will bind to another antibody
...
Stopping the energy transfer and gives a lower
emission
...
Relevant to different g-pathways
...
Detections in cAMP are also used in
this example, but utilising luminescence rather than fluorescence
...
Now there is a shift in these levels
(assuming increase), this increase in cAMP activates the kinase
...
For this

to occur there must be a source of phosphate groups (e
...
from ATP)
...

Increase in cAMP leads to a decrease of ATP
...

-

Luciferin is a luminescent compound
...
Light can only be
seen if ATP is used, however if this pathway is activated the ATP levels will decrease
...


All look at type 2 receptor activation
...

Fluorescence and luminescence comparison
-

Both are excitation pathways

-

Fluorescence uses the excitation of electrons to a higher energy level
...

Can give a higher background noise which must be calculated for
...
It can be difficult to
determine between excitation and emission wavelengths and there can be
interference from other fluorophores
...


-

Luminescence is a chemical process which usually involves an enzyme
...

Gives a slower rate of excited states, giving lower light intensities
...


-

Both can use small samples and give accurate readings compared to other assays,
such as radio-ligand assays
...
Tag-lite is a ligand binding assay which analyses the
competition between a fluorescent ligand and a compound that is not tagged
...
Majority of these are based of
FRET
...


Shows the surface receptors which are specific binding sites, which are designed to bind to a
ligand
...

Some ligands will also bind elsewhere
...
This could disrupt the fluorescence signal
...
The
total binding curve represents the sum of non-specific binding and specific binding
...
This is then taken into account and
allows the specific binding curve to be seen
...


Equation for calculation of specific binding
Specific binding = total binding – non-specific binding
KD determination
Multi-well plate contains a fix concentration of cells in each well and is incubated
...

Adding an additional non-labelled ligand is added and designed to interact with specific
binding sites
...
If this is present in
excess, it will displace the fluorescent ligand from specific binding sites
...

This method also gives a signal for total binding
...

Data interpretation
Need to plot specific binding signal
...


-

Using this the ratio of acceptor to donor emission signals can be calculated
...

Ratio = signal at 520/emission 620nm x 104
Plot this ratio signal vs the concentration of fluorescent ligand
...


-

This can be used to determine the dissociation constant (KD)
...
Half of

max signal is the signal at which half are occupied
...
This is KD
The lower the KD value, the stronger the affinity
...

A type of plot which can be used is a Scatchard plot which shows the ratio of bound
and unbound ligands
...

Plot ratio of bound to unbound ligands against the signal for the bound ligand
...
Fluorescence spectroscopy can be used as there is a fixed
concentration of a fluorescent ligand and a fixed concentration of cells
...

This shows a different binding curve
...

From this curve inhibitory concentration (IC50) can be determined
...
So, if is maximum response the IC50 will be 50% of
this
...


This is linked to the Cheng-Prusoff equation for receptor binding
This equation converts the IC50 value to a more universal constant of K i
...

To calculate this the dissociation constant of the labelled ligand must be known
...

-

Smaller the Ki value, the greater the affinity

-

If Ki value is smaller than KD, it can be assumed that the competitor has a stronger
affinity for the receptor than the fluorescent ligand
...
From this the specific binding signal is determined and increasing
amounts of agonist are added
...
This is done by plotting a
series of IC50 curves, which allows for comparison
...


It can be seen how concentration effects the inhibition effect
...
This is because of nonspecific binding
...

There is a relationship between the inhibition constant (Ki) and IC50
...
Whereas the IC50 is an indicator of the strength of inhibition
...

1
...
Un-competitive inhibition – binds to receptor/ligand complexes, limiting the
activity of the complex (IC50>Ki)
3
...
Therefore,
this takes into account different binding sites
...
Positive cooperativity
- Binding of the ligand molecule increases the receptors affinity
Oxygen binding to haemoglobin, haemoglobin has 4 subunits each with a binding
site
...
This approves the overall affinity
...
Negative cooperativity
- Binding of the ligand decreases the receptors affinity
Using the hill plot can help to determine what kind of binding is occurring along with the
dissociation constant
...
To
look at a competitor, increasing concentration of a competitor can be added
...
Must also have a control cell
which contains cells expressing receptors and a fixed concentration of a labelled
ligand
...

As increasing concentrations of test ligand (competitor) are added a decrease in
response is seen
...

(obtained from this plot)

-

-

Looking at the interaction of the labelled ligand (agonist) interacting with the
receptors
...

Physiological response (biological response) is the behaviour of the agonist
...

From the biological response KD for the labelled ligand can be calculated
...
This ligand has been added to all of the
wells
...
If a ligand is added in excess, it will displace the
tagged ligand except those bound to non-specific binding sites
...

Specific = total – non-specific

Rt = cell with no competitor present
𝐵
[𝐿]
=
[𝐿] + 𝐾𝑑
𝑅𝑇
Remember:
𝐿𝑜𝑔 (

𝐵
) = 𝐿𝑜𝑔[𝐿] − 𝐿𝑜𝑔𝐾𝐷
𝑅𝑇 − 𝐵

Hill plot:

-

Ratio of bound to unbound vs concentration of the ligand
...
(if log ration = 0 the antilog value is 1,
so if the ratio value is 1 this indicates a 50:50 split, so at equilibrium 50% of the
receptors are occupied)
X intercept = -LogKD

-

First calculate specific binding
Calculate log B/Rt-b
Calculate log[L] competitor values
Plot to give intercept which determines the dissociation constant of the unlabelled
ligand (competitor)

Can also get the hill coefficient from the hill plot: this is the gradient

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