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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.
Endothelin Receptor-Effector Coupling and Signaling in Cardiac Cells
Published in Malcolm J. Lewis, Ajay M. Shah, Endothelial Modulation of Cardiac Function, 2020
A practical response to worries about degradation is to include PMSF and leupeptin in physiologic buffer solutions. Adsorption, a related problem, may be minimized by use of siliconized surfaces. The inclusion of serum albumin (BSA, 1 mg/ml) seems to reduce the ravages of both adsorption and degradation. Hilal-Dandan et al. (1992) report that the inclusion of leupeptin (10 μg/ml) extended the linearity of inositol phosphate production by isolated ventricular myocytes.
Using a Recombinant Metagenomic Lipase for Enantiomeric Separation of Pharmaceutically Important Drug Intermediates
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Rakesh Kumar, Uttam Chand Banerjee, Jagdeep Kaur
Since the active site has been proposed to contain a conserved serine, PMSF was used as an inhibitor. To our surprise, PMSF did not inhibit the LipR1 lipase activity (Table 3.3). We observed temperature dependent inhibition with PMSF. At 55°C, LipR1 protein loses its 40–50% activity even with 1 mM of PMSF. Lipases have been shown to remain active in presence of PMSF in some cases (Li and Zhang, 2005; Soliman et al., 2007). It is very likely that active site serine is somehow inaccessible to PMSF yet at higher temperature some conformational changes in the protein makes the catalytic serine accessible to the inhibitor. Contrary to this DEPC, a histidine modifier inhibited the enzyme activity at very low concentration suggesting the easy accessibility of the catalytic histidine residue to DEPC. Eserine, an esterase’s inhibitor did not affect the enzyme activity, which suggested that it is a true lipase.
Exploring the mechanisms of action of Zengye decoction (ZYD) against Sjogren’s syndrome (SS) using network pharmacology and animal experiment
Published in Pharmaceutical Biology, 2023
Jiake Yu, Shuying Wang, Jie Yang, Wuxinrui Huang, Beikang Tang, Weijun Peng, Jing Tian
TNF-α, IL-1β, IL-6, and IL-17 enzyme-linked immunosorbent assay (ELISA) kits was purchased from Elabscience, China. Phenylmethylsulfonyl fluoride (PMSF) was purchased from Aladdin, China. A phosphatase inhibitor, radioimmunoprecipitation assay (RIPA) lysis buffer, and bicinchoninic acid (BCA) protein assay kit were ordered from Beyotime Biotechnology, China. Antibodies, including GAPDH, Bax, Bcl-2, Caspase-3 (CASP3), HRP-labeled rabbit anti-sheep IgG, and HRP-labeled rabbit anti-rats IgG, were purchased from Wuhan Sanyin Biotechnology Co., Ltd., China, and Wuhan Doctor De Biological Engineering Co., Ltd., China. An ECL substrate fluid was obtained from Beijing Plilai Gene Technology Co., Ltd., China. An X-ray photograph was purchased from Ruike Medical Equipment Co., Ltd., China. A developing and fixing kit was purchased from Tianjin Hanzhong Photography Materials Factory, China.
m-Calpain is released from striatal synaptosomes
Published in International Journal of Neuroscience, 2023
Nina Pestereva, Irina Ivleva, Alexander Zubov, Maria Tikhomirova, Marina Karpenko
Rat striatal synaptosomes were prepared on discontinuous Percoll gradients [9]. In brief, striatum from five rats was carefully dissected, homogenized in ice-cold 320 mM sucrose, 0.5 mM EDTA and 20 mM Tris–HCl (pH 7.4), and centrifuged for 5 min at 1,000 g, and the resulting supernatants were spun for 30 min at 15,000 g. The resulting pellet was gently resuspended in the same solution, layered onto a three-step gradient composed of 3, 10 and 23% Percoll, and centrifuged at 25,000 g for 20 min. The interface between the 10 and 23% layers containing the synaptosomal-enriched fraction was removed, added to 10 volumes of calcium-free Krebs–Ringer buffer containing 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM D(+)-glucose and 20 mM Hepes-NaOH at a pH of 7.4, and saturated with O2/CO2 mixture (95:5). The final synaptosomal pellet was resuspended in a Krebs–Ringer buffer at a total protein concentration of 1 mg/ml. Aliquots of synaptosomes were incubated at 37 °C for the indicated time in the presence of different concentrations of CaCl2 or EDTA. After incubation, the reaction tubes were centrifuged at 4 °C for 1 min at 15,000 g. PMSF (final concentration, 1 mM) was added to the supernatant, which was additionally centrifuged for 5 min at 15,000 g to remove tissue remains.
The BCL2/BAX/ROS pathway is involved in the inhibitory effect of astragaloside IV on pyroptosis in human umbilical vein endothelial cells
Published in Pharmaceutical Biology, 2022
Yi Su, Xin Yin, Xin Huang, Qianqian Guo, Mingyuan Ma, Liheng Guo
Mitochondrial proteins were extracted with the Cell Mitochondria Isolation Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China), following the manufacturer’s instructions. The HUVECs were cultured in 75 mm culture vessels with 2 × 107 cells in each vessel; then, they were incubated with different treatments for 24 h at 37 °C. Following incubation, the cells were collected using a trypsin–EDTA solution and centrifuged at 200 rpm at room temperature for 5 min. The cells were washed with pre-cooled PBS and centrifuged at 600 rpm at 4 °C for 5 min. The mitochondrial separation reagent containing phenylmethylsulfonyl fluoride (PMSF) was used to resuspend the cells, setting them on ice for 15 min. The cell walls were disrupted by ultrasound at 20 W, 10 s/time, until the trypan blue staining of the cells was ≥50%. The homogenized cells were centrifuged at 600 rpm at 4 °C for 10 min. The supernatant was centrifuged at 11,000 rpm at 4 °C for 10 min again. Mitochondrial lysate containing PMSF was resuspended.