Research methods in the study of endocrinology (1).pdf

Research methods in the study of endocrinology Endocrinology Methods Following the discovery of a hormone, endocrinology studies usually include the following investigations: 1. Identifying source (cells, tissue(s)) which produces the hormone 2. Structure determination: peptide hormone, primary aa s...

Research methods in the study of endocrinology Endocrinology Methods Following the discovery of a hormone, endocrinology studies usually include the following investigations: 1. Identifying source (cells, tissue(s)) which produces the hormone 2. Structure determination: peptide hormone, primary aa sequence, biological activity 3. 4. 5. 6. 7. Biosynthesis Control of secretion Circulation and metabolism Biological actions and roles Mechanisms of action Surgical Methods ◼ ◼ In animals Surgical removal of endocrine gland or tissue allows assessment of physiological alterations. Atrophy of target issue/organ ◆ Change in blood or urinary levels of certain metabolites or electrolytes ◆ ◼ First endocrine experiment (Berthold experiment): castration of cockerels (young male chicken) caused failure of developing their combs and wattles, and did not show male behaviour. Arnold Adolph Berthold 1803 - 1861 Surgical Methods A castrato (Italian, plural: castrati) is a type of classical male singing voice equivalent to that of a soprano, mezzo-soprano, or contralto. The voice is produced by castration of the singer before puberty, or it occurs in one who, due to an endocrinological condition, never reaches sexual maturity. Castration before puberty (or in its early stages) prevents a boy's larynx from being transformed by the normal physiological events of puberty. As a result, the vocal range of prepubescence (shared by both sexes) is largely retained, and the voice develops into adulthood in a unique way. Prepubescent castration for this purpose diminished greatly in the late 18th century and was made illegal in the Papal States, the last to prohibit them, in 1870. Farinelli is a 1994 internationally co-produced biographical drama film It centers on the life and career of the 18th-century Italian opera singer Carlo Broschi, known as Farinelli, considered the greatest castrato singer of all time. Farinelli Il Castrato 1994 Trailer HD - YouTube Tissue extraction and purification Crude extracts of endocrine tissue were used to determine whether these materials could replace the surgically removed endocrine gland Further purification of hormone from tissue extract is done Best and Banting experiments using dogs Sir Frederick Grant Banting 1891 –1941 Charles Herbert Best 1899 – 1978 Michael Bliss Glory Enough for All 1988 movie.depicting the discovery and isolation of insulin by Banting and Best. Use of antibodies to study hormones Proteins can be purified, quantitated and localized by highly specific antibodies Histological – Cytological studies Immunohistochemistry: Antibodies for a peptide or protein hormone are bound to a fluorescent dye and used to identify hormone producing cells. Insulin/pancreas Growth hormone/A. Pituitary Autoradiography Tissue is injected with a radioactively labelled hormone which selectively concentrates in particular target tissues. The tissue can be sliced and photographed, the radioactively labelled regions show as darkened spots on the film and show which structures have taken up the hormone in the largest amount. Immunological neutralization of hormone activity An antibody is prepared against a peptide hormone It is injected into an intact animal Ab neutralizes the biological activity of the endocrine hormone Bioassays: Study the action of hormones on living cells, tissues or organs In vitro (example: insulin action in muscle cells) In vivo Bioassays need not come from the same species. For example the 'rabbit test' (or Friedman test) for human pregnancy tested for the presence of human chorionic gonadotropin (hCG). This hormone is released from the implantation site on the wall of the uterus wall and prevents the menstrual cycle and can be measured in urine. Before hCG could be reliably detected in urine, a simple solution was to inject a sample of the urine into a female rabbit. If hCG were present, then the rabbit's ovaries would form ovarian structures that would not normallyform unless an egg had been fertilised. Bioassays:In vitro (example: insulin action in muscle cells) Glucose Transport Assay *radioactive substance (3H- glucose) Control * 3H-G 3H-G * Insulin Resveratrol * 3H-G 3H-G 3H-G 3H-G Cell 3H-G 3H-G 3H-G Cell Beta- Counter Prepare Cell Lysate Pharmacological Methods Determine mechanisms of hormone secretion and hormone action Actinomycine D – inhibits transcription Cytochalasin (B/D) microfilament Other inhibitors Cycloheximide (inhibits protein synthesis / translation) Radioisotope Studies Hormone radiolabeling animal studies to determine Half-life Tissue distribution Excretion rate Metabolism Solid Phase Immunoassay attachment of Ab 1 to support Ab 1 polymer support(polyvinylchloride) addition of sample washing to remove unbound molecules protein of interest addition of radiolabeled Ab 2 washing to remove unbound Ab2 radioactive label 125 I 125 I Ab 2 Enzyme-linked Immunosorbent assay (ELISA) Fluorescent product Nonfluorescent substrate enzyme protein of interest (Alcaline phosphatase) Ab 2 Ab 1 polymer support Example: pregnancy test: immunoassaying urine for humanchorionic gonadotropin (hCG) Immunoprecipitation of proteins Immunoprecipitation of proteins Very powerful when monoclonal antibody for the specific protein is available Protocol Protein sample antibody buffer (specific conditions for Ab-Ag interaction) incubate o/n at 4oC or 2-3 h at RT Add resin (protein A sepharose beads incubate, centrifuge dissolve pellet in appropriate buffer check the protein of interest (SDS-PAGE), use positive control Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting (measure protein expression/activation) Separates proteins according to their size Rapid Sensitive Capable of high degree of resolution Protein electrophoresis apparatus Western blotting 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Assemble apparatus Preparation of separating/stacking gel (note: acrylamide is very toxic) Load samples, molecular weight markers, protein positive controls Electrophoresis Transfer (blotting) to a membrane Blocking Incubation with primary Ab. Detection: secondary Ab (radioactive label, enzyme / ECL reaction) Autoradiogram Densitometry scanning Western blotting mixture of proteins==> solution with sodium dodecyl sulfate (SDS) SDS: anionic detergent ==> disrupts all noncovalent interactions Mercaptoethanol or dithiothreitol (DTD) ==> reduce disulfide bonds SDS binds to proteins (1 SDS / 2 AA) SDS-protein complex ==> net negative charge (proportional to protein mass,native charge render insignificant) electrophoresis ==> small proteins move rapidly through the gel *large proteins stay at the top SDS-PAGE (PolyAcrylamide Gel Electrophoresis) SDS-PAGE (PolyAcrylamide Gel Electrophoresis) Western blotting a b c a b c Enhanced chemiluminensent (ECL ) reaction oxidized product HRP: horseradish peroxidase secondary Ab HRP peracid primaryAb 2 H2O O2 2+ Lumimol + Enhancer light detection Summary of Western blotting 1. Assemble apparatus 2. Preparation of separating/stacking gel (note: acrylamide is very toxic) 3. Load samples, The protein sample you want to examine has been prepared ahead of time with SDS buffer. You also load molecular weight markers and protein positive controls. 4. Gel Electrophoresis 5. Transfer proteins (blotting) from the gel to a membrane 6. Blocking of the membrane with a solution that used dry milk or BSA. This step in the procedure aims to block the non-specific sites to prevent non specific binding of the Ab (next step) 7. Incubation with primary Ab. 8. Incubation with secondary Ab this will allows the detection of the protein as the secondary Ab has radioactive label (past) or is attached to an enzyme that when incubated with the substrate emits light (ECL reaction). ( today) 9. Autoradiogram (dark room, place film on the top of membrane, develop film, look at the bands) 10. Densitometry scanning ( measure the intensity of each band, this corresponds to the protein level, the darker the band the higher the levels of the protein) Blood [Hormone] measurements Blood [Hormone] very small: difficult to measure Radioimmunoassay: sensitive method for measuring [hormone] The Nobel Prize in Physiology or Medicine 1977 "for the development of radioimmunoassays of peptide hormones" Rosalyn Yalow Veterans Administration Hospital Bronx, NY, USA 1921 – 2011 Radioimmunoassay (RIA) Technique: A mixture is prepared containing radioactive hormone and antibody against this hormone A known amount of unlabeled hormone is added to the mixture. The labelled and unlabeled hormone compete for the binding sites on the antibodies. *With increasing concentrations of the unlabeled hormone, more radioactive hormones are displaced from the antibodies. The mixture is centrifuged, separating the hormoneantibody complexes from the free hormones. Radioimmunoassay (RIA) Radioactivity of both the supernatant (free hormone) and the precipitate (antibody bound) is measured. A ratio of bound to free hormone is determined for that concentration. By repeating the procedure with varying concentrations of the unlabeled hormone, and by plotting the ratio of bound to free hormone against the concentrations of the unlabeled hormone a standard binding curve is determined. The concentration of a hormone in a blood sample can than be determined by finding its ratio of bound to free hormone and comparing it with a standard curve from which the hormone concentration can be read. Radioimmunoassay (RIA) H Ab H H H test tube Hormone ‘cold” *H Ab Ab *H Ab Ab *H Ab Radioactive hormone “hot” antibody Radioimmunoassay (RIA) Radioimmunoassay (RIA) *H *H H *H *H Ab H *HAb *H Centrifuge (spin) *H H H H H *HAb *HAb *HAb Count radioactivity small number of counts ==> [H] high high number of counts ==> [H] low Ab radioactivity Radioimmunoassay standard curve Count radioactivity: small number of counts (cpm) ==> [H] high High number of counts (cpm) ==> [H] low Hormone (cold) concentration / [H] RIA Ab H *H Ab Ab H *H *H H Radioactive hormone H Ab Ab Ab hormone antibody *H H *H Ab *H *H Ab H H Ab H Ab *H Mix *HAb *H H H H Ab *H Ab time for equilibration H *HAb radioactivity Radioimmunoassay standard curve Count radioactivity: small number of counts (cpm) ==> [H] high High number of counts (cpm) ==> [H] low Hormone (cold) concentration / [H] How is insulin (a protein hormone) produced today to be used for the treatment of individuals with Type I diabetes? Type 1 Diabetes Injecting Insulin Recombinant DNA technology DNA cloning Process by which a plasmid is used to import recombinant DNA into a host cell for cloning. Plasmid: Plasmids are small circles of DNA found in bacteria cells, separate from the bacterial chromosome and smaller than it. They are able to pass readily from one cell to another, even when the cells are clearly from different species, far apart on the evolutionary scale. Consequently, plasmids can be used as vectors, permitting the reproduction of a foreign DNA by using the bacterial replicating system. A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for the same restriction endonuclease. The plasmid and the foreign DNA are cut by EcoRI producing intermediates with sticky and complementary ends. Those two intermediates recombine by base-pairing and are linked by the action of DNA ligase. A new plasmid containing the foreign DNA as an insert is obtained. A few mismatches occur, producing an undesirable recombinant. The new plasmid can be introduced into bacterial cells that can produce many copies of the inserted DNA. This technique is called DNA cloning. 1993 Nobel Prize in Chemistry Kary Mullis for his invention of the polymerase chain reaction (PCR) method. PCR turns the job over to the very biomolecules that nature uses for copying DNA: two "primers" that flag the beginning and end of the DNA stretch to be copied; an enzyme called polymerase that walks along the segment of DNA, reading its code and assembling a copy; and a pile of DNA building blocks that the polymerase needs to make that copy. Kary Mullis 1944 – 2019 As he wrote in Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents and a source of heat. The DNA sample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials.The DNA may come from a hospital tissue specimen, from a single human hair, from a drop of dried blood at the scene of a crime, from the tissues of a mummified brain or from a 40,000-year-old wooly mammoth frozen in a glacier." The Polymerase Chain Reaction is a technique used very extensively in molecular genetics, forensics and other fields to greatly amplify a specific DNA sequence. Kary Mullis who received the 1993 Nobel Prize in Physiology and Medicine for the discovery invented it in 1983. A PCR amplification reaction includes: the source DNA containing the sequence of interest (as few as 1 copy), two unique single-stranded DNA primers that bracket the desired sequence, deoxyribonucleotides (the building blocks of DNA), and Taq DNA polymerase (from the thermophilic organism Thermus aquaticus). The reaction is cycled between three temperatures that facilitate the construction of new copies of the desired DNA sequence. At 95?C, the double stranded source (or template) DNA is denatured into single strands. Then, at 55oC, the primers bind (anneal) to complimentary sequences on the template DNA that bracket the sequence to be amplified. Last, at 72oC, the Taq DNA polymerase adds deoxyribonucleotides in sequence to the primer as it builds a complementary strand to the template DNA. This cycle is repeated until a very large number of copies are obtained. The reaction is usually carried out in a machine called a thermal cycler that quickly changes the temperature of the reaction mixture between the three necessary temperatures. A good graphic illustration of the process can be found at http://www.accessexcellence.org/AB/GG/polymerase.html. Polymerase Chain reaction (PCR) Thermocycler apparatus (PCR machine) Design oligonucleic primers : complimentary to two different regions of the DNA. Tube: Amplification buffer Mixture of dNTPs Primer 1 Primer 2 DNA Taq DNA polymerase Polymerase Chain reaction (PCR) taq DNA polymerase from thermus aquaticus is heat stable enzyme (94oC), carries out synthesis of complementary strand of DNA from 5’ to 3’ direction by the primer extension reaction. Primers Designed so that primer 1 directs the synthesis of DNA towards the other, primer 2, and vice versa. Result synthesis of the region of DNA flanked by the two primers. PCR Purpose of PCR is to make a large number of copies of a gene. The procedure is performed in a machine which heats and cools the PCR mixture. PCR can be divided into three main steps which are repeated a number of times (around 30). 1. Denaturation (at 94°C) - the double stranded DNA is opened 2. Annealing (at 54°C) – single stranded primers constantly form and break ionic bonds with the single stranded template. Polymerase attaches on more stable bonds and starts coping the template. This makes the ionic bond stronger and prevents it from breaking. 3. Extension (at 72°C) – Weak ionic bonds are broken in this temperature and so only primers with strong ionic bonds with the template remain attached. Polymerase works optimally at this temperature, and continues to add bases where some have already been added. Polymerase Chain reaction (PCR) In vitro amplification of specific DNA sequences using appropriate primers Rapid Sensitive Inexpensive Use Analysis of DNA RNA genetic diagnosis Detection of mutations Genetic engineering Molecular Techniques Structure-activity studies Site-directed mutagenesis Can be used to synthesize a mutant receptor with a changed aa sequence (deletion of a region) Examine hormone receptor function Determine sequence responsible for: Ligand (hormone) binding Signal transduction Genetic Engineering Transgenic animals Foreign DNA inserted into the mammalian genome Foreign DNA → microinjection → production of fertilized cells → microinjection → implantation of the cells into the uterus of the mother → transgenic mouse E.g.. Growth Hormone over expressing mice Transgenic mice Genes responsible for particular traits or disease susceptibility are chosen and extracted. Next they are injected into fertilized mouse eggs. Embryos are implanted in the uterus of a surrogate mother. The selected genes will be expressed by some of the offspring. Since the first gene transfers into mice were successfully executed in 1980, transgenic mice have allowed researchers to observe experimentally what happens to an entire organism during the progression of a disease. Transgenic mice have become models for studying human diseases and their treatments. Normal mouse Growth Hormone overexpressing mouse Growth Hormone overexpressing salmon Genetic manipulations New advances in molecular biology have meant that specific genetic manipulations can now be carried out, the commonest being the insertion of genes (transgenic) or the removal of genes (knockout) encoding a hormone or a hormone receptor. If such mutants are bred with normal animals (wild-type) then their offspring will be heterozygous (have one copy) for that trait. If mutants breed with mutants animals then their offspring will be homozygous for the mutation. The offspring of these homozygous individuals will all carry the genetic mutation. Fetal Tissue transplant – Stem Cell Research Animal Models: human fetal brain cells transplanted to monkeys E.g. Parkinson’s Disease Fetal Islet cell transplant restore insulin production Examples of exam questions 1. What is polymerase chain reaction (PCR)? Describe PCR in detail. Make sure to describe all the steps and details for a full mark. 2. What is Radioimmunoassay (RIA)? Describe RIA in detail. Make sure to describe all the steps and details for a full mark. 3. What is western blotting. Describe western blotting. Make sure to describe all the steps for a full mark. 4. Currently recombinant DNA technology is used to produce Human insulin used to treat Type 1 Diabetes patients. Describe the basic steps involved in human insulin production. The end