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Noninvasive Screening for Malignant Hyperthermia by Means of the Lymphocyte Test
Published in S. Tsuyoshi Ohnishi, Tomoko Ohnishi, Malignant Hyperthermia, 1994
Beverley A. Britt, Amira Klip, Peter J. O’Brien, Barbara I. Kalow
Fluorescent assay buffer is composed of 10 mM HEPES, 10 mM glucose, 140 mM NaCl, 3 mM KCl, and 1 mM MgCl2, pH 7.3. This solution is passed through a filter with a 0.22-μm pore diameter to remove any particulate matter that might interfere with fluorescence determinations. Following this, 5 mM TPEN, a heavy metal chelator, is added to the buffer. This addition has been made only in studies utilizing Indo 1, but could also be employed if Quin 2 is utilized as the fluorescent indicator. Measurements of the buffer, when using Quin 2, are made at a wavelength of 339 nm for excitation (3 nm-slit width) and 485 nm for emission (15-nm slit width). When using Indo 1, measurements are made at a wavelength of 331 nm for excitation (3-nm slit width) and 485 nm for emission (15-nm slit width). All measurements are carried out at room temperature. To calibrate the baseline, fluorescence is read first in the presence of the buffer alone in a fluorescence quartz (not plastic) cuvette placed over a specially adapted constant magnetic stirrer within the spectrofluorometer. Then 20 μ1 of the indicator-loaded lymphocytes are added to 1.0 ml of the assay buffer and the fluorescence measurement is repeated. CaCl2 1.0 M is added to the mixture and fluorescence is also measured at this time to make certain that the lymphocytes are not leaky. A small increase in fluorescence (about 10%) is expected under these conditions.
Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2+
Published in Drug and Chemical Toxicology, 2019
Masamichi Ae, Naohiro Imura, Tomoko Inubushi, Shin Abe, Bekki Yusuke, Mayumi Sugimoto, Norio Kamemura
Increases in intracellular Zn+2 levels are known to result in increases in oxidative stress (Chimeddorj et al.2013, Slepchenko et al.2017). In addition, Matsui et al. (2010) reported that an increase in intracellular Zn+2 levels induces cell death and sensitivity to oxidative stress and that TPEN and DTPA completely attenuate elevations in cytotoxicity induced by increases in intracellular Zn+2 levels. Our results showed that biphenyl at concentrations above 30 μM significantly increased the intensity of FluoZin-3 fluorescence in isolated rat thymocytes. In addition, TPEN, but not DTPA, inhibited the increase in intracellular Zn+2 levels, cell lethality, and sensitivity to oxidative stress (Figures 3 and 4). These results showed that the increase in intracellular Zn+2 levels induced by biphenyl is dependent on internal Zn2+. Moreover, we confirmed that the cytotoxic effects of biphenyl were caused by the increase in intracellular Zn+2 levels.
Low molecular mass natural and synthetic inhibitors of snake venom metalloproteinases
Published in Toxin Reviews, 2018
Lina María Preciado, Jaime Andrés Pereañez
Specific zing chelating agents such as N, N, N′, N′-Tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) (Figure 3C), diethylene triamine pentaacetic acid (DTPA) (Figure 3D) and tetraethylthiuram disulfide (TTD) (Figure 3E) have been evaluated in the inhibition of hemorrhage and myotoxicity induced by E. carinatus (ECV) venom. These compounds completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; however, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and BM surrounding the blood vessels in mouse skin sections and prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. Subsequent studies confirmed, by spectral and docking experiments, that TPEN has high affinity for Zn2+. This specificity explains the potent inhibition of ECV metalloproteinases (ECVMPs) in vitro (IC50: 6.7 mM) (Nanjaraj Urs et al., 2015). However, the major limitation of TPEN is its toxicity as it may sequester physiologically important Zn2+ containing enzymes at higher doses.
Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes
Published in Drug and Chemical Toxicology, 2019
Honoka Wada, Keisuke Oyama, Risa Kamae, Toshiya Masuda, Kaori Kanemaru, Kumio Yokoigawa, Yasuo Oyama
Cellular and membrane parameters were measured using a flow cytometer equipped with an argon laser (CytoACE-150; JASCO, Tokyo, Japan) and fluorescent probes, as previously described (Matsui et al. 2008). The fluorescence was analyzed by JASCO operating system software (Version 3.06; JASCO, Tokyo, Japan). Under experimental conditions, no fluorescence from the reagents used was observed, except for the fluorescent probes. The excitation wavelength for all fluorescent probes used in this study was 488 nm, while emissions wavelengths were 530 ± 20 nm for FluoZin-3 and 5-CMF, and 600 ± 20 nm for PI. To assess cell lethality, PI was added to the cell suspensions to a final concentration of 5 µM. Because PI stains dead cells, the measurement of PI fluorescence provides information on cell lethality. Fluorescence was measured using a flow cytometer 2 min after adding PI. FluoZin-3-AM was used to monitor changes in the intracellular Zn2+ levels ([Zn2+]i) (Gee et al.2002). The cells were treated with 1 µM FluoZin-3-AM for 60 min prior to any fluorescence measurements. FluoZin-3 fluorescence was monitored from the cells without PI fluorescence. 5-CMF-DA was used to estimate the cellular content of glutathione ([GSH]i) in rat thymocytes (Chikahisa et al.1996). The 5-CMF fluorescence was measured 30 min after adding 1 μM 5-CMF-DA because it attains peak intensity within 30 min after application. Living cells that were not stained with PI were examined for 5-CMF fluorescence. Zn2+-free condition was prepared by adding Zn2+ chelator (DTPA or TPEN) to the cell suspension 10 min at least before the experiments. Because DTPA is not membrane-permeable, DTPA chelates extracellular Zn2+. Membrane-permeable TPEN can chelate intracellular Zn2+.