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Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
In this study, we compared the secretion pattern and cellular localization of wild-type TTR, 7 TTR variants and their monomeric mutants (M-TTRs) using TTR secretion assay and immunofluorescence analysis. Five of these TTR variants (CNS-selective amyloidoses, D18G and A25T TTR; systemic amyloidoses, V30M, E54K and L55P TTR) are disease-associated and are termed ‘amyloidogenic TTRs’, whereas two of the variants we used, T119M and R104H, have no known pathogenic consequences and are termed ‘non-amyloidogenic TTRs.’ Here, we report that D18G TTR and amyloidogenic M-TTRs were barely secreted into the cell media and that they were retained in the ER, but the amyloidogenic TTR variants and the non-amyloidogenic M-TTRs were efficiently secreted from the cells. Moreover, amyloidogenic M-TTRs and D18G TTR are degraded by ERAD. These findings raise the possibility that ER quality control strictly monitors the folded state of TTR monomer level. Because most amyloidogenic TTR variants have moderate
Structure-Function Elucidation of Flavonoids by Modern Technologies
Published in Dilip Ghosh, Pulok K. Mukherjee, Natural Medicines, 2019
Ritu Varshney, Neeladrisingha Das, Rutusmita Mishra, Partha Roy
Pancreatic β-cell degeneration and dysfunction plays an important role in the pathogenesis of type 1 and type 2 diabetes. Insulin, which is secreted from β-cells, is a key mediator of glucose metabolism. Thus, deficiency of insulin synthesis and secretion or lack of insulin sensitivity leads to decreased glucose tolerance which results in diabetes. Insulin is synthesized from preproinsulin which is further processed to proinsulin. Proinsulin is then processed to insulin and C-peptide and stored in secretory granules until release on stimulation. Insulin biosynthesis is regulated at both transcriptional as well as translation level. Thus, flavonoids can play a role in increasing insulin synthesis or secretion or both the activities, which can be tested using suitable assays. Some of the phytochemicals such as genistein (Skelin et al. 2010; Fu and Liu 2009), quercetin (Youl et al. 2010), berberine, ginsenoside, curcumin (Dai et al. 2013), and epigallocatechin-3-gallate (Youl et al. 2010) have been reported to be effective in augmenting insulin secretory activity of pancreatic β-cells. To elucidate the insulin synthesis activity of any phytochemical, insulin promoter activity assay can also be performed over and above transcriptional analysis of insulin gene, while for insulin secretion assay, the secreted insulin level in cell cultures can be quantified by insulin ELISA (enzyme-linked immunosorbent assay; Jang et al. 2013). For in vitro insulin synthesis and secretory assays, the most widely used pancreatic β-cell lines are RIN-m5F, RIN-5F, MIN6, HIT-T15, INS-1 and β-TC-6 (Labriola et al. 2009).
Anti-inflammatory, expectorant, and antitussive properties of Kyeongok-go in ICR mice
Published in Pharmaceutical Biology, 2021
Jin-Ryul Hu, Chul-Jong Jung, Seong-Min Ku, Dae-Hwa Jung, Khawaja Muhammad Imran Bashir, Sae-Kwang Ku, Jae-Suk Choi
Mucosal secretions were calculated using the tracheal phenol red secretion assay. A single intraperitoneal injection of 5% phenol red (Junsei Chemical Co. Ltd., Tokyo, Japan) in saline (w/v; 10 mL/kg) was administered 30 min after the last treatment with a test substance (day 11). Thirty min post phenol red injection, all mice were killed by cervical dislocation without damaging the trachea, gross images were acquired to monitor redness on body surface. After isolation from adjacent organs, the trachea was removed from the main stem bronchi and the thyroid cartilage and ultrasonicated for 15 min (Branson Ultrasonics, Danbury, CT). After sonication, 1 mL of 5% NaHCO3 was add to the normal saline, and the optical density of the prepared tracheal lavage fluid (TLF) was measured at 546 nm (Tecan, Männedorf, Switzerland; Engler and Szelenyi 1984; Zhang et al. 2009; Wang et al. 2012).
NPM-ALK-reactive T-cell responses in children and adolescents with NPM-ALK positive anaplastic large cell lymphoma
Published in OncoImmunology, 2019
Vijay Kumar Singh, Sebastian Werner, Simone Schwalm, Volker Lennerz, Stephanie Ruf, Serena Stadler, Holger Hackstein, Alfred Reiter, Thomas Wölfel, Christine Damm-Welk, Wilhelm Woessmann
The IFN-γ secretion assay technique allows identifying antigen-specific IFN-γ secreting T-cells in a mixed population of cells.58 NPM-ALK-peptide specific IFN-γ-secreting T-cells were detected by using the IFN-γ secretion assay kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Responder T-cells, frozen before, from peptide pool-reactive donation were thawed and cultured overnight in AIM-V medium supplemented with 5% human serum and 5 IU/ml IL-2. Then, the T-cells were seeded in a 48-well plate (1 – 2x1059/well) in AIM-Vstim and stimulated at a 10:1 ratio with autologous DCs pulsed either with the peptide pools or with individual peptides of a positive-pool. After 12 hours incubation in the presence or absence of antigen, T-cells from each 48-well were processed according to the manufacturer’s guidelines and resuspended in the flow cytometry-buffer. IFN-γ secreting cells were immediately analyzed by the FACS Calibur system (BD).
A benzimidazole-based ruthenium(IV) complex inhibits Pseudomonas aeruginosa biofilm formation by interacting with siderophores and the cell envelope, and inducing oxidative stress
Published in Biofouling, 2019
Grzegorz Czerwonka, Dawid Gmiter, Anna Guzy, Patrycja Rogala, Agnieszka Jabłońska-Wawrzycka, Andrzej Borkowski, Tomasz Cłapa, Dorota Narożna, Paweł Kowalczyk, Marcin Syczewski, Marcin Drabik, Magdalena Dańczuk, Wiesław Kaca
Pyoverdine determination was performed in TSB medium. Measurement of pyoverdine fluorescence was performed at the excitation wavelength λ = 398 nm and the emission wavelength λ = 455 nm as described previously (Imperi et al. 2009). Experiments were carried out in triplicate in 96-well black flat-bottomed microtiter plates (Greiner Monroe, NC, USA) for 24 h in cell-free culture medium using an Infinite M200PRO (Tecan, Männedorf, Switzerland). The assay was performed with ligand (2-hydroxymethylbenzimadazole), the ruthenium(IV) complex and streptomycin as a positive control, and TSB medium with inoculated bacteria as a negative control. The siderophore secretion assay was conducted as three independent repeats. The results obtained were not disturbed by the emission band characteristic of the ruthenium(IV) complex (see Supplemental material Table S1).