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Peptidases and Peptides at the Blood-Brain Barrier
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Janet Brownlees, Carvell Williams
This membrane-bound metalloenzyme is found in many different tissues, including the microvessels of mammalian brain.133,134 It has broad substrate specificity and in brain tissue has been particularly implicated in the inactivation of enkephalins. Cloning studies in rat,135 mouse136 and human133 suggest that it is a typical ectoenzyme with one membrane-spanning region. The cerebrovascular enzyme was shown to degrade Met-enkephalin by removal of the N-terminal Tyr1 residue (Km = 91.3 μM).133 Some longer opioid peptides e.g. β–endorphin, were resistant to the enzyme. It is inhibited by the metal chelating agents, EDTA and 1,10-phenanthroline, and by amastatin (IC50 0.2 μM), bestatin (IC50 10 μM) and less effectively by puromycin (IC50 90 μM). Several peptides were shown to be effective competitive inhibitors of Met-enkephalin degradation by cerebrovascular aminopeptidase M including neurotensin (Ki = 20.4 μM) and substance P (Ki = 7.9 μM). Pardridge et al.138 report that analogues of somatostatin are rapidly sequestered by cerebral microvessels and then metabolized by an aminopeptidase that may be aminopeptidase N.
Tonic Influences on Baroreceptor Reflex Sensitivity By Endogenous Neurotensin in the Nucleus Tractus Solitarii
Published in I. Robin A. Barraco, Nucleus of the Solitary Tract, 2019
Julie Y.H. Chan, Samuel H.H. Chan
Along with being a specific arginyl aminopeptidase and leucine aminopeptidase inhibitor,16-18 bestatin may also be a potent blocker of alanyl and leucyl aminopeptidase.16 Based on these properties and presently available data on amino acid sequences and peptide degradation, it follows that the neuropeptides most likely to be protected by this degradative enzyme blocker may at least include, albeit not exclusively, neurotensin, angiotensin III, substance P, and somatostatin.
Resveratrol Blunts Mitochondrial Loss in Slow and Mixed Skeletal Muscle Phenotypes of Non-Human Primates following a Long-Term High Fat/Sugar Diet
Published in Journal of Dietary Supplements, 2023
Jon-Philippe K. Hyatt, Rafael de Cabo, Julie A. Mattison
To ensure homogenous total protein isolation, SOL and PLT muscle sample chunks from the approximate muscle belly were mounted on cork and cut cross-sectionally using a cryostat (CM1950; Leica, Buffalo Grove, IL). Approximately 30–80 15 µm-thick cross sections were acquired, which equated to ∼50–100 mg sampled from each muscle. Samples were then bead homogenized for 2.5 min at 3,000 rpm in 10-volume ice-cold buffer (pH 7.8) containing 50 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Triton-X, and a 1% v/v final concentration of a protease and phosphatase inhibitor cocktail (PPC1010; Sigma, St. Louis, MO) containing 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin hydrochloride, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide, leupeptin, pepstatin A, cantharidin, (–)-p-Bromolevamisole oxalate, and calyculin A. After homogenization, the samples were centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was transferred to clean tubes in a biological safety cabinet, and then frozen at −80 °C.
Time matters – in vitro cellular disposition kinetics help rationalizing cellular potency disconnects
Published in Xenobiotica, 2022
Birk Poller, Sophie Werner, Norbert Domange, Lina Mettler, Richard R. Stein, Jacqueline Loretan, Markus Wartmann, Bernard Faller, Felix Huth
The transcellular permeability assay in MDCK-KO cells has been described in detail by Huth et al. (Huth et al. 2021). In brief, cells were seeded on Transwell 96-well plate inserts (Corning, Tewksbury, MA), at a density of 1.5 × 105 cells/mL and grown for 4 days. Each compound was dosed in triplicate in the apical donor compartment at a final concentration of 10 µM in Hanks’ balanced salt solution (HBSS) at pH 7.4 containing 10 mM HEPES, 100 nM bafilomycin and 0.02% w/v bovine serum albumin (BSA). The same assay buffer was used in the basolateral acceptor compartment with the exception of a higher BSA concentration (5%). Cells were incubated with the compounds for 2 h at 37 °C, and flux was measured in the apical-to-basolateral direction. Bestatin was included in each well to monitor integrity of the cell monolayer and only wells with a Papp <1.5 × 10−6 cm/s were accepted. Drug concentrations in the donor and acceptor compartments were measured by liquid chromatography mass spectrometry (LC-MS), using a six-point calibration curve, glyburide as an analytical internal standard (see details below).
Recent advances in IAP-based PROTACs (SNIPERs) as potential therapeutic agents
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Chao Wang, Yujing Zhang, Lingyu Shi, Shanbo Yang, Jing Chang, Yingjie Zhong, Qian Li, Dongming Xing
Statistics were performed using data extracted from PubMed and PROTAC-DB to determine the frequency of various IAP ligands used in SNIPERs (Figure 5) 37,38. The LCL-161 derivative was the most commonly used, with approximately 31% of SNIPERs employing its structure. It was closely followed by bestatin, with lower occurrence of MV1 derivatives and IAP ligand 4 at 10% and 9%, respectively. Other IAP ligands were less common, with less than 3% representation.