Module 4 Flashcards
(32 cards)
What was Berthold’s experiment?
1 pt
- 1st recorder endocrine experiment by Professor Arnold A. Berthold of Gottingen in 1849
- Did a series of tests on roosters while he was curator of the local zoo
- Based on observations of prepubertal castration of male chicks (used to make capons, more tender meat):
- reduced secondary male appearance (combs + waffles)
- failed to exhibit male behaviour - Thought it was dependent on neural connections
What did Berthold discover in his experiment?
2 pt
- on autopsy the transplanted testis had reestablished a blood supply, but not a neural supply
- reimplanted or transplanted testis was also approximately 2X larger than normal
What were Berthold’s conclusions?
3 pt
The testis could maintain such function by:
1. activation or transofrmation of factors in blood to active agents, or
2. removal of inhibitory substances in the blood, or
3. release of factor (hormone) into circulation
How was the existence of a hormone discovered?
1 pt
- First critical experiment demonstrating the existence of a hormone reported in 1902
- Knew that acid in partially digested foodstimulated pancreatic secretion after it entered the small intestine
- introduced acid into an isolated (denervated) but vascularized section of jejunum - cause similar flow of pancreatic juice
- supported view that the control of pancreatic enzymes was mediated by bloodborne stimulation
- extracts of jejunal epithelium produced similar effects
What would endocrinologists want to know about a particular hormone?
10 pt
- Source distribution (organ, tissue, cell)
- Structure determination and synthesis
- Biosynthesis
- Control of synthesis
- Cellular mechanisms of secretion
- Circulation and metabolism
- Biological actions and roles
- Mechanism of action
- Pathophysiological aspects
- Comparative endocrinology
What are the hormone concentrations used in endocrinology studies?
2 pt
Physiological levels
* ranges of ug/ml - pg/ml in blood or extracellular fluid
Pharmacological levels
* commonly in the mg/ml range
* produce circulating levels in excess of natural levels
* effects of pharmacologic doses of hormones may be different from responses to physiological levels and may be therapeutically useful (i.e. glucocorticoid - inflammation)
* more risk of toxic response
Examples of hormones (or their analogues) that are administered as medication/theraputics?
2 pt
- insulin
- birth control
What are the experimental test systems?
5 pt
- Whole animal
- Isolated perfused organs
- Tissue slices
- Isolated cells in culture
- Subcellular fractions
How does a whole animal model work?
3pt
The classical methods used to study endocrine tissues are whole animal studies in which organs are removed and replaced.
* Remove organ
- Surgical
- Chemical
- Deafferentation
* Replace organ
- Ectopic site replacement
- Inject extracts of gland
* Monitor physiological responses
What observations are taken after removal?
4 pt
What effects could be observed following removal of the endocrine organ/tissue?
* atrophy of target organs
* changes in blood/urine conc of hormones
Surgical removal
* adrenalectomy, thyroidectomy
* but hard to do in complex organs
Can use drugs (pancreatic islet ablation, alloxan)
* Can target and inactivate specific cells
Can also remove nervous stimulation to an organ = deafferentation (eg vagus nerve and gastrin secretion in the stomach)
What observations are made after replacement and examples of replacements?
3 pt
- Replacing lost tissue (surgery or disease state) through transplants, administering extracts of the tissue, administering actual purified hormone or analog
- Crude extracts -> isolated hormones -> pure preparation
- Ex. insulin, estrogen (from pregnant mares)
What pharmacological manipulations are used on the endocrine system?
3 pt
- Agonists
- Antagonists
- Inhibitor
What is an agonist, antagonist, and inhibitor?
3 pt
- Agonists: bind to hormone receptor and mimic action
- Antagonist: block/inhibit action of hormone at receptor
- Inhibitor: block action of hormone downstream of receptor
What are examples of agonists, antagonists, and inhibitors?
4 pt
- inhibitors of RNA and protein synthesis (including hormones)
- Thiouracil -> inhibits TH synthesis - hormone receptor antagonistsand agonists
- cyproterone acetate -> testosterone receptor antagonist - phosphodiesterase inhibitors
- elevate cAMP levels by inhibiting degradation of cAMP - metabolic inhibitors
- inhibit glucose uptake or ATP formation
What are some methods in endocrinology?
4 pt
- Radioimmunoassay (RIA)
- Immunohistochemistry
- Measuring gene expression
- Polymerase Chain Reaction
- In situ Hybridization - Genetic Engineering
- Transgenic Assays
- Knockout Animals
What would be the method used for each of these questions:
1. Where is the receptor/protein located in the tissue/cell?
2. What is the concentration of hormone or receptor in circulation?
3. What is the expression (mRNA levels) of specific hormone-responsive genes in tissues?
4. What is the FUNCTION of a hormone in the whole organism?
4 pt
- Immunohistochemistry
- RIA
- Measuring gene expression
- Genetic Engineering
What is Histological-Cytological Studies?
1 pt
Histology allows us to tell the cytological (cellular) characteristics of endocrine tissue
* Cells that are hypertrphic (enlarged)
* Cells that are atrophic (diminished)
Significant changes from the normal in organ or cell size may provide clues to an underlying pathophysiology (or the functional changes that accompany a particular syndrome or disease)
What is histology?
1pt
Histological stains can tell us basic components of the cell
* Hematoxylin (purple): basic dye interacts with acidic parts (phosphoric acid of DNA + RNA)
* Eosin (pink): acidic dye interacts with basic parts (cytoplasm)
What is immunohistochemistry?
1 pt
Immunological methods = use specific antibodies to detect and label specific antigens (molecular targets of immune mediators)
* Hormones, receptors, cell signalling molecules, transcription factors, other proteins
Immunohistochemistry = characterise the specific distribution (i.e. where are they?) of a target protein in tissues, cells or parts of cells
Antibodies are specific and will bind to a specific portion (epitope) of the target molecule
* Antibody prepared by injecting hormones or receptors obtained from one species into another species
What is radioimmunoassay?
1 pt
- Immunological-based assay -> similar to immunohistochemistry
- Allows the detection and quantification of minute concentrations of hormones in blood or other fluids
- Procedure depends on the binding of the specific antibody to the radiolabeled antigen (hormone); this is the same as receptor-hormone binding kinetics convered in Module 2
*[H] + [Ab] <—-> *[H][Ab]
^[H] +[H] + [Ab] <—-> [H][Ab] +^[H][Ab]
What is the principle of radioimmunoassay?
1 pt
An unlabeled hormone [H] when added to a reaction mixture will compete with the radiolabeled hormone *[H] for binding to the antibody
What is the analysis of gene expression in endocrine systems?
2 pt
The process by which a gene gets turned on in a cell to make RNA and protein = gene expression
* GE is measured by levels RNA, or the protein made from the RNA
* Hormones induce effects by changing the levels of mRNA or protein in target tissue
* Hormone genes themselves are also subject to regulation (eg. peptide-protein hormones)
Expression of certain genes can be up-regulated or down-regulated
What is Polymerase Chain Reaction?
1 pt
- PCR is a powerful tool to measure gene expression
- All of the extracted RNA is reverse transcribed into its DNA complement using an enzyme (reverse transcriptase)
- Specific primers designed against a specific sequence are used to amplify the message in the form of its copy DNA (cDNA)
What is PCR Analysis?
1 pt
- Signal intensities are compared to those of control genes, thus detecting relative changes in expression levels
- Fluorescence (real-time PCR)
- Standard technique that is easily employable in most laboratories
