China
Africa
Explore chapters and articles related to this topic
Role of Beta Adrenoceptors in the Control of Myocardial O2 Supply/Consumption Heterogeneity
Published in Samuel Sideman, Rafael Beyar, Analysis and Simulation of the Cardiac System — Ischemia, 2020
Harvey R. Weiss, Mary E. Upsher
Hearts were cut on a band saw at −20°C. The left ventricular free wall was then cut into 15 roughly equal pieces (approximately 1 to 2 g). The location of each was recorded and all samples were divided into subendocardial and subepicardial regions. Each of the 30 tissue samples were weighed, placed in a tube with 5 ml of 25 mM HEPES (pH 7.5) containing 178 mM NaCl and 6.3 mM MgCl2, and then dropped into liquid nitrogen. These samples were maintained in the frozen state until used for β-adrenoceptor studies. Each frozen tissue sample was warmed to 0°C, washed free of blood, and homogenized in 10 to 20 volumes of ice-cold HEPES. The homogenate obtained was passed through cheesecloth and centrifuged at 20,000 X g for 10 min at 4°C. The supernatant was discarded and the pellet was resuspended in the appropriate volume of HEPES to give 1.0 mg of protein per milliliter.
Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
For each sample, 50 µL of resuspended cell pellet is added to Pico Prias (PerkinElmer) or equivalent vials containing 3 mL of scintillation cocktail (e.g. Optiphase Hisafe II; PerkinElmer). The rest of the cell homogenate can be stored at −20°C and used for determination of protein concentration, e.g. by the Lowry method. As controls, 50 µL of NaOH is added to 3 mL of scintillation cocktail (negative control), and 50 µL of 14C-propionate medium is added to 3 mL of scintillation cocktail (total input control). Close the vials, mix well and use a beta-counter (e.g. Tri-Carb C1900TR; PerkinElmer) to determine radioactive counts per minute (cpm) for each sample at least twice.
Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
Mammary glands were obtained from lactating rabbits or pigs and stored at -20°C until use. The glands were homogenized with 4 vol (per weight of the gland) of 0.25 M sucrose for 30 to 45 sec in a Polytron homogenizer attached with a PT 10 head at a speed setting of 7. The homogenate was filtered through three layers of cheesecloth and centrifuged at 8000 × g for 15 min at 4°C. The supernatant was centrifuged at 100,000 × g for 75 min at 4°C. The pellet was suspended in 25 mM Tris-HCl (pH 7.4)-10 mM MgCl2-l mM phenylmethylsulfonyl fluoride (PMSF) (Tris-HCl-Mg-PMSF). The crude microsome suspension was kept frozen at -20°C until use.
Veratramine ameliorates pain symptoms in rats with diabetic peripheral neuropathy by inhibiting activation of the SIGMAR1-NMDAR pathway
Published in Pharmaceutical Biology, 2022
Yu Zhang, Guangyao Ye, Yuebo Chen, Chaoxu Sheng, Jianlin Wang, Lingsi Kong, Liyong Yuan, Chunyan Lin
The protein expression levels of SIGMAR1, NMDAR, and p-NMDAR (Ser896) were detected by Western blotting (WB). L4-L5 spinal cord tissues were collected from rats. Then, RIPA lysis buffer and phosphatase inhibitors were added to the tissue, and the tissue was homogenised by a tissue fragmentation instrument (Qiagen, Germany) on ice. The samples were centrifuged at 10,000g at 4 °C for 15 min, and the supernatant was collected. The homogenate was then denatured at 100 °C for 5 min. The denatured protein was added to a 12.5% SDS–PAGE gel, and electrophoresis was terminated by running out the separation gel until bromophenol blue at 90 V. The protein was then transferred to PVDF (Millipore, Germany). Next, 3% BSA was used for blocking overnight at 4 °C. After blocking was completed, the membranes were washed in TBST three times, and rabbit anti-rat SIGMAR1 (1:1000, cat no. 15168-1-AP, Proteintech), NMDAR (1:1000, cat no. 5704S, Cell Signaling Technology), p-NMDAR (Ser896, cat no. ARG51606, Arigobio), or GAPDH (1:1000, 2118, CST) were added and incubated at room temperature for 2 h. After washing with TBST, goat anti-rabbit IgG-HRP secondary antibody (1:2000) was added and incubated at room temperature for 2 h. ECL chemiluminescence agent (Sharebio, China) was used for luminescence, and imaging was performed using the UVP Imaging System (ChemiDoc-It Imaging System, USA). Relative protein expression was calculated using ImageJ software using GAPDH as an internal reference protein.
MiADMSA abrogates sodium tungstate-induced oxidative stress in rats
Published in Drug and Chemical Toxicology, 2022
Sherry Sachdeva, Ankita Sharma, S. J. S. Flora
MiADMSA was selected at 50 mg/kg on the basis of previous published literature which proved it to be the minimum effective dose with least side effects (Flora et al. 2012). MiADMSA was dissolved in normal saline and administered to rats, also, an equal volume of saline was given to normal control and tungstate control animals. The body weight of rats was recorded every alternate day to monitor any possible change in body weight during dosing. The animals wre sacrificed 48 h after the last dosing to allow sufficient time for complete absorption of the dose material. After the completion of the treatment schedule. Euthanasia was administered using Isoflurane inhalant followed by gentle cervical dislocation. Rats were then placed in a closed glass chamber where high levels of anesthetic gas (3%) were introduced. 1 ml of blood samples were collected from unconsciousness animals in ethylenediamine tetraacetic acid (EDTA) anticoagulant tubes from the retro-orbital vein by a glass capillary tube puncture. Liver, kidney and spleen were collected, washed with cold normal saline, blotted and all the extraneous materials were removed. The homogenate was used for the estimation of various biochemical parameters. Tissues were stored at −20 °C till further analysis.
Spectroscopic observations of β-eudesmol binding to human cytochrome P450 isoforms 3A4 and 1A2, but not to isoforms 2C9, 2C19, and 2D6
Published in Xenobiotica, 2022
Dawid Krenc, Kesara Na-Bangchang
The frozen lysozyme digest was placed on ice and per 1.0 mL digest was added 10 μL of 4 U/mL aprotinin (Cayman Chemicals, Ann Arbour, MI), 10 μL of 1 mg/mL bestatin (EMD Millipore, Burlington, MA), 10 μL of 1 mg/mL leupeptin (Sigma-Aldrich), and 10 μL of 100 mM isopropanolic phenylmethylsulfonyl fluoride (Sigma-Aldrich). The lysozyme digest was briefly thawed in water (room temperature) and sonicated with a VibraCell VC750 probe (Sonics, Newtown, CT) in an ice-salt bath as follows: 9 s at 30% amplitude, 9 s pause, for 10 min. The supernatant was transferred to a 15-mL tube for centrifugation at 10,000 g (4 °C) for 10 min (Sorvall Legend RT + Centrifuge, Thermo Scientific), followed by another transfer and centrifugation at 14,000 g (4 °C) for 10 min. The volume was recorded (‘S14000’ volume), and the cell supernatant was further centrifuged at 225,000 g (rmax.) (4 °C) for 2 or 3 h (TH-641 rotor, Sorvall WX Ultra 100, Thermo Scientific). The ultracentrifuge supernatant was decanted and the pellet was submerged in 1.0 mL cold filtered 1 × TES buffer (50 mM Tris-acetate pH 7.5, 0.25 mM EDTA, and 250 mM sucrose). For homogenisation, a total of no more than half the recorded S14,000 volume of cold, filtered 1 × TES buffer was added to the pellet. This was transferred to an ice-cooled Dounce homogeniser of 5.0 mL capacity and subjected to about 10 swift up-and-down strokes. The homogenate was aliquoted in 0.6-mL tubes and stored at −20 °C.