Calcitonin-human

The process for the manufacture of human calcitonin in pure form from C-cell rich medulla... 

FIGURE 2.2 Binding and dose-response curves for human calcitonin on human calcitonin receptors type 2. (a) Dose-response curves for microphysiometry responses to human calcitonin in HEK cells (open circles) and binding in membranes from HEK cells (displacement of [,25I]-human calcitonin). Data from [1]. (b) Regression of microphysiometry responses to human calcitonin (ordinates) upon human calcitonin fractional receptor occupancy (abscissae). Dotted line shows a direct correlation between receptor occupancy and cellular response. 

FIGURE 2.11 Receptor-occupancy curves for activation of human calcitonin type 2 receptors by the agonist human calcitonin. Ordinates (response as a fraction of the maximal response to human calcitonin). Abscissae (fractional receptor occupancy by human calcitonin). Curves shown for receptors transfected into three cell types human embryonic kidney cells (HEK), Chinese hamster ovary cells (CHO), and Xenopus laevis melanophores. It can be seen that the different cell types lead to differing amplification factors for the conversion from agonist receptor occupancy to tissue response. 

FIGURE 3.11 Constitutive activity in melanophores expressing hCTR2 receptor, (a) Basal melanophore activity, (b) Effect of transfection with human cDNA for human calcitonin receptors (16 j-ig/ml). (c) Concentration response curve for cDNA for human calcitonin receptors (abscissae as log scale) and constitutive activity. Data redrawn from [27]. 

Chen, W.-J., Armour, S., Way, J., Chen, G., Watson, C., Irving, P., Cobb, J., Kadwell, S., Beaumont, K., Rimele, T., and Kenakin, T. P. (1997). Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors. Mol. Pharmacol. 52 1164-1175. 

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

FIGURE 5.6 Calcitonin receptor responses, (a) Real-time melanin dispersion (reduced light transmittance) caused by agonist activation (with human calcitonin) of transfected human calcitonin receptors type II in melanophores. Responses to 0.1 nM (filled circles) and lOnM (open circles) human calcitonin, (c) Dose-response curves to calcitonin in melanophores (open circles) and HEK 293 cells, indicating calcium transient responses (filled circles). 

FIGURE 5.11 Microphysiometry responses to 1 nM human calcitonin, (a) Responses obtained from HEK 293 cells stably transfected with low levels of human calcitonin receptor (68 pM/mg protein). Response is sustained, (b) Response from HEK 293 cells stably transfected with high levels of receptor (30,000 pM/mg protein). Data redrawn from [8],... 

Armour, S. L., Foord, S., Kenakin, T., and Chen, W.-J. (1999). Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor. J. Pharmacol. Toxicol. Meth. 42 217—224. 

This concept is illustrated by the example shown in Table 11.1. Shown are three replicate pEC50 values for the agonist human calcitonin obtained from two types of cells wild-type HEK 293 cells and HEK 293 cell enriched with Gas-protein. The respective pECS0 values are 7.47 0.15 and 8.18 0.21. The question is Do these two estimates come from the same population That is, is there a statistically significant difference between the sensitivity of cells enriched and not enriched with GffiS-protein to human calcitonin To go further toward answering this question... 

In fact, a measure of the degree of confidence can be gained from the t calculation. Shown in Appendix A are columns for greater degrees of confidence. The value for df = 4 for a 98% confidence level is 3.747 and it can be seen that the experimentally calculated value is also greater than this value. Therefore, the level of confidence that these samples came from different populations is raised to 98%. However, the level of confidence in believing that these two samples came from separate populations does not extend to 99% (t = 4.604). Therefore, at the 98% confidence level this analysis indicates that the potency of human calcitonin is effectively increased by enrichment of G -protein in the cell. 

FIGURE 11.3 One-way ANOVA (analysis of variance). One-way analysis of variance of basal rates of metabolism in melanophores (as measured by spontaneous dispersion of pigment due to G,.-protein activation) for four experiments. Cells were transiently transfected with cDNA for human calcitonin receptor (8 j-ig/ml) on four separate occasions to induce constitutive receptor activity. The means of the four basal readings for the cells for each experiment (see Table 11.4) are shown in the histogram (with standard errors). The one-way analysis of variance is used to determine whether there is a significant effect of test occasion (any one of the four experiments is different with respect to level of constitutive activity). 

Methodology. Several radioimmunoassays of human calcitonin (hCT) have been developed In the past 5 years (18-20). Their greatest utility has been In the definitive diagnosis of patients with medullary carcinoma of the thyroid gland (MTC) and, recently. In Identifying family members of these patients who have occult MTC. 

Hill, C. S. Jr. "Immunoassay of Human Calcitonin Clinical Measurement, Relation to Serum Calcium and Studies in Patients With Medullary Carcinoma". N. Engl. J. Med. (1970), 283. 890-895. 

M39. Morris, H. R., Panico, M., Etienne, T., Tippinss, J., Girgis, I., and Maclntre, I., Isolation and characterization of human calcitonin gene-related peptide. Nature 308,746-748 (1984). 

Kanaori et al. [1.122] studied the mechanism of formation, and association of human calcitonin (hCT) fibrils using NMR. hCT associates and precipitates during storage in aqueous solution. The freeze dried hCT and its behavior is described. 

Reches, M., Porat, Y., and Gazit, E. (2002). Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin./. Biol. Chem. 277, 35475-35480. 

Fig. 2. Electron micrographs highlighting the polymorphism of amyloid fibrils. (A) A single human calcitonin protofibril with a diameter of 4 nm (adapted from Bauer et al., 1995). (B) Different morphologies present in a transthyretin fibril preparation. Black arrowheads show oligomers of different sizes, the black arrow points to a 9- to 10-nm-wide fibril, and the white arrowhead marks an 4-nm-wide fibril (adapted from Cardoso et al., 2002). (C-F) Human amylin fibril ribbons (adapted from Goldsbury et al., 1997). (C) A single 5-nm-wide protofibril. (D-F) Ribbons containing two (D), three (E), or five (F) 5-nm-wide protofibrils. (G) A twisted ribbon made of four 5-nm-wide protofibril subunits of Api-40 (adapted from Goldsbury et al., 2000b). Scale bar, 50 nm (A-G).

Bauer, H. H., Aebi, U., Haner, M., Hermann, R., Muller, M., and Merkle, H. P. (1995). Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin./. Struct. Biol. 115, 1-15. 

P Sieber, B Riniker, M Brugger, B Kamber, W Rittel. 255. Human Calcitonin. VI. The synthesis of calcitonin M. (side-chain alkylation) Helv ChimActa 53, 2135, 1970. 

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