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    Comparison of ELISA and RIA methods to quantify arginine vasopressin hormone levels in cell culture
    (Springer, 2023) Turkmen, Merve Ozcan; Karaduman, Tugce; Mergen, Hatice
    The radioimmunoassay (RIA) method is widely used to determine the levels of arginine vasopressin (AVP) in studies, especially in cell culture studies. However, there are many difficulties and disadvantages in performing this method. Therefore, this study aimed to assess the comparison between enzyme-linked immunosorbent assay (ELISA) and RIA methods by using the Bland-Altman statistical method and to investigate whether the commonly used RIA can be replaced by the ELISA method for measurement of AVP levels in cell culture medium. For this purpose, wild-type (WT) and three different mutant AVP-NPII vectors were transiently transfected to mouse neuroblastoma (Neuro2A) cells and AVP secretion into the cell culture medium by transfected Neuro2A cells was determined by both RIA and ELISA methods. Following the use of the normalization method, Bland-Altman method, currently the most commonly used statistical method assessing comparison between two methods, was performed to assess agreement between two methods. The order of normalized AVP values obtained using the RIA method was as follows: WT (0.921 +/- 0.08), 207_209delGGC mutant (0.801 +/- 0.09), G45C mutant (0.508 +/- 0.10), and G88V mutant (0.497 +/- 0.12). The normalized AVP values obtained using the ELISA method were as follows: WT (0.865 +/- 0.12), 207_209delGGC mutant (0.704 +/- 0.13), G88V mutant (0.255 +/- 0.16), and G45C mutant (0.250 +/- 0.15). The order of AVP levels measured from each transfected cell using both methods was quite similar. According to the Bland-Altman method, there is an agreement between RIA and ELISA for measuring the AVP levels in cell culture. It can be recommended to apply ELISA instead of the RIA method for determinating AVP levels.
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    Functional analyses of three different mutations in the AVP-NPII gene causing familial neurohypophyseal diabetes insipidus
    (Springer, 2021) Turkmen, Merve Ozcan; Karaduman, Tugce; Tuncdemir, Beril Erdem; Unal, Mehmet Altay; Mergen, Hatice
    Purpose Familial neurohypophyseal diabetes insipidus (FNDI), a rare disorder, which is clinically characterized by polyuria and polydipsia, results from mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The aim of this study was to perform functional analyses of three different mutations (p.G45C, 207_209delGGC, and p.G88V) defined in the AVP-NPII gene of patients diagnosed with FNDI, which are not included in the literature. Methods For functional analysis studies, the relevant mutations were created using PCR-based site-directed mutagenesis and restriction fragment replacement strategy and expressed in Neuro2A cells. AVP secretion into the cell culture medium was determined by radioimmunoassay (RIA) analysis. Fluorescence imaging studies were conducted to determine the differences in the intracellular trafficking of wild-type (WT) and mutant AVP-NPII precursors. Molecular dynamics (MD) simulations were performed to determine the changing of the conformational properties of domains for both WT and 207-209delGGC mutant structures and dynamics behavior of residues. Results Reduced levels of AVP in the supernatant culture medium of p.G45C and p.G88V transfected cells compared to 207_209delGGC and WT cells were found. Fluorescence imaging studies showed that a substantial portion of the mutant p.G45C and p.G88V AVP-NPII precursors appeared to be located in the endoplasmic reticulum (ER), whereas 207_209delGGC and WT AVP-NPII precursors were distributed throughout the cytoplasm. Conclusions The mutations p.G45C and p.G88V cause a failure in the intracellular trafficking of mutant AVP-NPII precursors. However, 207_209delGGC mutation does not result in impaired cellular trafficking, probably due to not having any significant effect in processes such as the proper folding, gain of three-dimensional structure, or processing. These results will provide valuable information for understanding the influence of mutations on the function of the AVP precursor hormone and cellular trafficking. Therefore, this study will contribute to elucidate the mechanisms of the molecular pathology of AVP-NPII mutations.
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    Functional analysis of AQP2 mutants found in patients with diabetes insipidus
    (Springer, 2021) Karaduman, Tugce; Ozcan Turkmen, Merve; Ozer, Emel Saglar; Ergin, Bora; Saglam, Berk; Erdem Tuncdemir, Beril; Mergen, Hatice
    Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the reabsorption of water in the main collecting duct cells of kidneys. Mutations in AQP2 gene induce nephrogenic diabetes insipidus (NDI), a pathogenic condition involving the maintenance of body water homeostasis, leading to excess urine production. Several hypotheses in the literature were confirmed by clinical and experimental observations regarding the role of these mutations in the pathogenesis of NDI. In this study, three mutations (A45T, R85X, and A147T) identified in NDI patients were analyzed using stably transfected Madin-Darby canine kidney (MDCK) cells and Xenopus laevis (X. laevis) oocyte expression system compared to the wt-AQP2. According to our study, the A45T-AQP2 protein is characterized by reduced half-life and targeting defects, therefore this mutant protein can be retained in the endoplasmic reticulum (ER). The mutation also causes the formation of a nonfunctional water channel. The R85X-AQP2 protein was not detected in immunoblot analysis, but the results of water permeability test data prove that this mutant protein cannot form a functional water channel. Despite its features such as partial false targeting and reduced half-life, A147T-AQP2 protein forms a semi-functional water channel. In a conclusion, studies on the intracellular functions of mutant water channels are important because these studies elucidate the mechanisms leading to disease phenotype and support development of pharmacological strategies related to pathological mechanisms.