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44561
Methods in endocrinology
Description
Endocrinology Mind Map on Methods in endocrinology, created by maisie_oj on 12/04/2013.
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endocrinology
endocrinology
Mind Map by
maisie_oj
, updated more than 1 year ago
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Created by
maisie_oj
over 11 years ago
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Resource summary
Methods in endocrinology
Areas of study
Glands
Target tissues/cells
General methods
Surgical manipulation - e.g. removal/replacement of a gland
Physiological alterations
In target tissue
Loss of activity
Chnages in blood composition?
e.g. castration or adrenalectomy
reversed by hormone replacement therapy
Tissue extract preparation
Histological methods - ID target cells (immunohistochemistry, staining)
Cytological characteristics
Hypertrophic - enlarged tissue (not number of cells)
Organelles - number present, size etc.
Dilated ER/golgi = increased activity
Atrophy (wasting of tissue)
Hyperplasia (increased number of cells)
Disease status
Stains (of chemical components in cell)
Glycoproteins
Glycogen
Lipid
Can indicate function
Gene expression
Pharmacological - mimic/inhibit activity
Anima models
General considerations
Source
Structure and synthesis
Control of secretion and mechanism
Circulation and metabolism
Actions and roles
Pathophysiological roles
Comparative endocrinology
Studying the endocrine control of a physiological process
For hormones secreted by a ductless gland
Remove the gland
observe effects
Is effect restored after gland extract?
Purified/synthetic hormone induec same effect?
Secretion - mechanism and control
ID receptors on target cells
Many receptors found - some are referred to as orphans as their ligands are not known
Gross observation of tissue
Anatomical location
Size
Vascularisation / innervation
Pathophysiologies - e.g. goitre - indicating a thyroid problem
Immunocytochemistry
Techniques
Immunocytochemistry
Can involve
Ab's
Very specific
For peptide/protein hormones
Conjugated
With fluorescent tags/enzymes that produce a coloured prduct
Method
Tissue sample prepared
Incubated in primary Ab for specfic cell protein
Incubated with secondary Ab carrying a fluorescent tag
Visualisation
Dependent on method
Immunofluorescence (with fluorescent Ab's)
Immunoperoxidase (if enzyme-conjugated)
Fluorescent in situ hybridisation (FISH)
Detects gene amplification using a fluorescently labeleld gene probe
Aids treatment selection in breast cancer (HER2 amplification? - herceptin appropriate?)
Radioisotopes
Considerations
Half life of isotope
Trace distribution throughout the body
e.g. using radioisotopic iodine in the diagnosis (and treatment) of hyperthyroidism
Autoradiography
Uptake and incorporation of isotope into cells metabolic pathways - e.g. nucleotide synthesis / glucose metabolism
Cellular site of action
Steroids (gene expression) - intracellular
peptide hormones - extracellular
Radioimmunoassay (RIA)
Sensitive and highly specific (due to use of Ab's)
In vitro assay used to determine levels of hormone in sample (e.g. blood)
Method
Known amount of antigen is made radioactive and mixed with known amount of Ab = hot antigen
Next a sample (from the patient) of unknown antigen concentration is added = cold antigen
The unbound cold antigen competes for the Ab and displaces bound hot antigen
the amount of displacement correlates to the amount of cold antigen in the sample
The solution containing the unbound antigens (both hot and cold) is then separated and its radioactivity measured (geiger counter)
The level of radiation is proportionate to the amount of displaced radioactive antigen
This can be compared to a standard curve of solution radiation against known amounts of cold antigen (done previously)
Can be affected by episodic/diurnal release
Tissue extracts
Crude extraxcts (preparation from tissue - not purified)
Replace excised tissue
Can be purified - i.e. hormonal supplements
Can be used as treatment (i.e. insulin)
Tissue extract can be analysed using subcellular fractionisation
Using differential centrifugation
Subcellular fractions separated based on density/size
Then tested for receptor/hormone of interest (determines cellular location)
Detecting protein or mRNA of interest
Nothern blot
Detects mRNA
Simliar to western blot
Radiolabelled/fluorescent RNA primer used intead of Ab
Western blot
Detects proteins
Extract all proteins (lysis)
give negative charge or denature
Run on SDS page
Produce Ab
Transfer SDS page to nitrocellulose
Incubate with primary antibody
Wash off excess Ab and incubate with secondary Ab (+ fluorescent tag)
Detect fluorescence
PCR
Amplification of short mRNA sequence (testing transcription level)
Convert mRNA from cell into cDNA using a reverse polymerase
Bioassays
Use of a biological response to detect presence of a hormone in serum/tissue extract
The level of response to a hormone can be measured and a standard produced for known concetrations of hormone
Patient serum containing an unknown concentration of the hormone can then be tested and compared against the standard curve
Structure-activity assays
Looking at the structure of a compound and comparing this to its funtion
Antagonist or agonist?
Use known amounts of a hormone to produce a dose response curve (standard curve) - forms a sigmoid curve due to ligand receptor kinetics
Repeat with test compound
Is max response reached quicker/at all?
Site-directed mutagensis
Induce genetic mutation
To detect functional domains
Can be tested for using a reporter assay
Constructed gene with a hormone response element, basic promotor region and reporter gene (e.g. beta-galactosidase or luciferase)
If mutation affects a functional domain of the hormone/hormone receptor (e.g. steroid hormone receptor) then there is no trasncription of the reporter
Visual result: beta-galactosidase expressing cells grow blue on X-gal medium; and luciferase produces light
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