Chapter 2 Methods to Study Bioregulation Copyright © 2013 Elsevier Inc. All rights reserved.

Chapter 2Chapter 2

Methods to Study BioregulationMethods to Study Bioregulation

Copyright © 2013 Elsevier Inc. All rights reserved.

Figure 2-1 Importance of initial controls. Comparison of data for animals taken at the beginning of the experiment (initial controls) allows one to determine whether the experimental treatment stimulated the animals (A) or simply maintained the initial conditions (B) while the “controls” declined.

2Copyright © 2013 Elsevier Inc. All rights reserved.

Figure 2-2 Dose–response relationships. (A) A system may respond to a threshold dose and then continue to show increased response with increasing dose until the process reaches a maximum. Additional bioregulator then produces no increased response over a wide range. (B) Relationship of optimal response to an “optimal” dose. A dose too low to produce a response is considered subthreshold. Doses above the “optimal” dose produce diminished responses and may indicate a toxicity to the tissue or animal. Alternatively, the bioregulator may reach a threshold that triggers accelerated removal of the bioregulator and hence a reduction in response at higher doses. Such doses often are considered to be “pharmacological,” although any level of a regulator that exceeds natural levels also could be termed “pharmacological.” Notice that when observing the response of a single dose you might not know whether you were above the optimal dose or below it. Consequently, when the data are not readily available, multiple doses should be employed to determine a useful dose–response relationship. (C) Species-specific or tissue-specific dose–responses. An “optimal” dose for one tissue or one animal might be either subthreshold or pharmacological for another. (D) J-shaped or U-shaped dose–response curves are common in biological systems when dealing with very low doses.


Figure 2-3 Diurnal pattern of plasma corticotropin (ACTH) and cortisol. More ACTH produced in the pituitary gland is released in the early morning than at other times of day. Because ACTH stimulates the adrenal cortex to secrete cortisol, the latter shows a matching rhythm in blood concentration trailing that of ACTH. (Adapted with permission from Vinson, G.P., “The Adrenal Cortex,” Prentice Hall, Upper Saddle River, NJ, 1993.)


Figure 2-4 Confocal laser microscopy. Application of confocal laser microscopy to the visualization of calcium movement in a single pituitary melanotrope cell. By vertically stacking successive images through the cell, a 3D image is created. Changes in fluorescence caused by calcium movement (red, high; blue, low) can be detected in multiple planes (X, Y, t) sliced through the cell. (Courtesy of Dr. Bruce Jenks, Department of Cellular Animal Physiology, Radboud University Nijmegen. From Koopman, W.J. et al., Biophysics Journal, 81, 57–65, 2001.)


Figure 2-5 Reconstruction from multiphoton and confocal microscopy. Elucidating the pattern and timing of pituitary LH (purple) and POMC (green) cells in the developing (A) and adult (B) mouse pituitary as reconstructed from multiphoton and confocal microscopy images. Panel C outlines the developmental sequence for cell appearance based upon the microscopic analysis. POMC cells first appear in the ventral anterior lobe (1) and then extend laterally and ventrally into the AL (2, 3). The first POMC cells in the intermediate lobe (melanotropes) appear next (4). LH cells then appear in the AL, first in the ventral AL (5) and then in the dorsal AL (6). Islands of melanotropes are found in the dorsal AL postnatally. (Reprinted with permission from Budry, L. et al., Proceedings of the National Academy of Sciences USA, 108, 12515–12520, 2011.)


Figure 2-6 Standard curve for radioimmunoassay (RIA). A standard curve is prepared by placing the same amount of antibody and radiolabeled (hot) hormone into every test tube and adding a different known amount of unlabeled (cold) hormone to each tube so that cold and hot hormone will compete for the same binding sites on the antibody. The greater the amount of cold hormone added, the lower will be the amount of radioactivity bound to the antibody. The antibody with its bound load of hot and cold hormone is precipitated from the solution and either the radioactivity of the supernatant or of the precipitate is counted. Thus, one can plot the percentage of hot hormone bound to the antibody against the amount of cold hormone in the solution to produce a standard relationship or what is called a standard curve. If an additional tube is prepared with the same amounts of antibody and hot hormone but with an unknown amount of cold hormone (such as might be present in a blood sample), one can determine the percentage of hot hormone bound (A) and extrapolate from the standard curve to estimate how much cold hormone (B) was present in the blood sample.


Figure 2-7 High-performance liquid chromatography (HPLC). A sample is applied to a separation column in a particular solvent. Additional solvents can be mixed and pumped through the separation column and, depending on the affinity of the sample components for the column and the solvents, the sample components will leave the column at different times and pass through some kind of detector. Some compounds, such as steroids, are best detected and quantified by their absorbance of ultraviolet light (UV), whereas others (e.g., catecholamines) can be quantified using electrochemical detectors. HPLC can be coupled to radiation detectors for identifying a labeled molecule or specific metabolic products of a radioactive precursor, and the radiation detector may be used in tandem with either UV or electrochemical detectors. Various fractions from the column may be recovered by using a fraction collecting device.


Figure 2-8 Immunocytochemistry. This method requires a pure source of antigen to make a specific antibody (primary antibody) in a mouse (or rabbit, etc.). Although the primary antibody usually is applied to sections of cells or tissues, this method also can be used on whole blocks of tissue. A secondary antibody is made against mouse immunoglobulins (for example, in a goat) to amplify the location of the primary antibody that is bound in situ to the antigen. The secondary antibody can be complexed to an enzyme, a fluorescing compound, or a radioactive marker for detecting the exact location of the antigen in a cell or tissue. This approach can be used for detecting the presence or absence of an antigen or to determine the number of reactive cells, etc. It also can be coupled with other techniques to estimate the intensity of the reaction and relate that to the quantity of antigen present.


Figure 2-9 Confocal laser microscopy identifies multiple antigens in a single cell. Phosphorylated STAT3 (signal transducer and activator of transcription 3) is upregulated in cells of the mouse hypothalamus expressing the neuronal marker HuD/C after intraperitoneal injection of the appetiteregulating hormone leptin. (From Frontini, A. et al., Brain Research, 1215, 105–115, 2008.)


Figure 2-10 “Specific” and “nonspecific” binding by receptors. Determination of saturable (receptor or “specific”) and nonsaturable (nonreceptor or “nonspecific”) binding of cortisol to a liver cytosolic preparation using 3H-cortisol as the ligand. Nonsaturable binding is not affected much by the amount of ligand because it has a high capacity. This is determined by adding a large excess of unlabeled cortisol in an attempt to displace the saturable receptors (specific binding) while not affecting the nonsaturable (nonspecific) binding. The difference between the binding observed with and without the excess of unlabeled ligand is defined as the saturable or specific binding. (Adapted from Chakraborti, P.K. and Weisbart, M., Canadian Journal of Zoology, 65, 2498–2503, 1987.)


Figure 2-11 Scatchard plotting method for receptor determination. The Scatchard plot is described in the text. (From Bolander, F.F., “Molecular Endocrinology,” Academic Press, San Diego, CA, 1989.)


Figure 2-12 Microarray hybridization. Basic protocol for determining large scale changes in gene expression using microarray hybridization. RNA is isolated from cells or tissues and reverse transcribed to cDNA. cDNA is then used to produce fluorescently labeled copies of cRNA that are hybridized on microarrays containing as many as 44,000 gene targets.


Figure 2-13 Chromatin immunoprecipitation (ChIP) assay. See text for explanation. (Adapted with permission from Collas, P. and Dahl, J.A., Frontiers in Bioscience, 13, 929–943, 2008.)


Figure 2-14 Transgenic animals. Use of Cre/loxP system for producing transgenic animals with gene expression deficits in selected target cells. (A) The pro-opiomelanocortin (POMC) gene cannot be expressed when loxP (locus of X-over P1) sites are present surrounding the stop sequence, despite the ubiquitous promoter that is active in many different cell types. When recombined with the gene encoding the DNA recombinase Cre, which is expressed only in special cell types because of the tissue-specific promoter, the loxP sites are deleted and the POMC sequence can be transcribed. (B) Transgenic mice expressing Cre in special cell types are mated with mice containing a construct in which the target gene is surrounded by loxP sites. Offspring will lack target gene expression in only those cells expressing Cre.


Figure 2-15 Methodology for identifying proteins using 2D gel electrophoresis followed by LC-MS/MS. See text for explanation.


Chapter 2 Methods to Study Bioregulation Copyright © 2013 Elsevier Inc. All rights reserved.

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