Carboxylesterase

Primary cultures of dermal fibroblasts were established by explantation from biopsies of neonatal foreskins, or from affected dorsal forearm of seven patients with SSc and four healthy controls [3] (link), [56] (link). Only patients with diffuse cutaneous SSc (dcSSc) who were not receiving treatment with corticosteroids were studied. Primary cell cultures were also established from the dorsal skin of newborn Smad3-null (Smad3^–/–) mice or wildtype (Smad3^+/+) littermates [30] (link). Unless otherwise indicated, all fibroblasts were maintained at 37°C in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% vitamins, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD), and studied between passages 4 and 8. When fibroblasts reached early confluence, fresh media containing TGF-ß (Genzyme, Framingham, MA) were added to the cultures. In selected experiments, cultures were pretreated with 15d-PGJ₂ (BioMol, Plymouth Meeting, PA), troglitazone (GlaxoSmithKline, King of Prussia, PA), rosiglitazone (Cayman Chemical, Ann Arbor, MI), interleukin-4 (IL-4) or platelet-derived growth factor (PDGF) (both from Sigma, St. Louis, MO) for indicated periods. In other experiments, the protein kinase inhibitors U0126 (Cell Signaling Technology, Beverly, MA), SB431542 or SB203580 (both from Sigma) at 10 µM, or SP600125 (Sigma) at 20 µM, were added to the cultures 60 min prior to TGF-ß. In experiments focusing on profibrotic responses in fibroblasts, TGF-β at 10 ng/ml, a concentration shown to be maximally effective and non-toxic in prior studies, was used. Cell viability was determined by Trypan blue dye exclusion.
Human peripheral blood macrophages were isolated from buffy coats from healthy donors (Lifesource, Glenview, IL) by Histopaque (Sigma) gradient centrifugation followed by countercurrent centrifugal elutriation (JE-6B, Beckman Coulter, Palo Alto, CA). Isolated monocytes were ≥90% pure as determined by morphology, non-specific esterase staining and CD14 expression by flow cytometry. Following adherence to plates for 60 min, monocytes differentiated into macrophages for seven days in Roswell Park Memorial Institute (RPMI) media containing 20% heat-inactivated FBS, L-glutamine, and penicillin and streptomycin, and used in experiments.

The docking study was performed using ezCADD [61 (link)] Smina [62 (link)] software (https://www.dxulab.org/software) (accessed on 1 August 2022). The ezCADD is a web-based CADD modeling environment where one of its applications is to simulate protein-ligand interactions. 2D/3D depictions were visualized using the BIOVIA discovery studio 2021 client software [63 ]. The simulation system was built on the crystal structures of PDB ID: 1AO6 [64 (link)], 1P0I [65 (link)], 1ACJ [66 (link)] and 1EVE [67 (link)], which were downloaded from the Protein Data Bank (https://www.rcsb.org (accessed on 1 August 2022)). These proteins were classified into antibodies against Human Serum Albumin (HAS) and Hydrolase (hydrolase carboxylic esterase and hydrolase inhibitor). The water molecules and ligands were removed from the PDB file using pymol [68 ] and polar hydrogen bonds were added [69 ]. Automatic cavity detection was performed using fpocket3 [70 (link)]. The minimum binding affinity values (kcal/mol) were calculated to evaluate the interactions between the docked ligands and the studied proteins.

FB₁ degradation by FumDO or FumDM was performed using an appropriate amount of diluted enzyme solution in 50 mmol/L sodium phosphate buffer (pH 6.5) containing FB₁ (5 µg/mL) at 40 °C for 10 min, and then the mixture was boiled for 10 min to terminate the reaction. One unit (U) of carboxylesterase activity was defined as the amount of enzyme required to degrade 1 µg FB₁ per minute at 40 °C and pH 6.5.
FB₁ detoxification activities of FumDO/FumDM in this study were detected by high-performance liquid chromatography (HPLC), which was performed on a Waters Alliance e2695 HPLC system with a 2475 fluorescence detector (Waters Co., Ltd., Milford, MA, USA). In order to add fluorescence detective signals onto FB₁, samples were derived by mixing 100 µL of enzyme reaction mixture with 400 µL of 50% acetonitrile aqueous solution and 500 µL of a o-phthaldialdehyde (OPA) derivative solution. Then, samples were filtered through a 0.22 µm nylon filter and stored in sampler vials for injection within 2 min. Separation was performed on a C18 column (5 µm particle size, 250 × 4.6 mm X-bridge, Waters Co., Ltd., Milford, MA, USA) with a flow rate of 1.0 mL/min at 40 °C and the injection volume was set at 50 µL. All samples were analyzed at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The mobile phase consisted of 0.1 mol/L ammonium formate—formic acid aqueous solution (A) and 100% chromatographic grade methanol (B). The gradient was as follows: 0.00 min 30% A, 5.00 min 28% A, 6.00 min 25% A, 11.00 min 22% A, 11.10 min 30% A, and 16.00 min 30% A. Waters ChemStation was employed for the LC system to acquire and analyze chromatographic data. The degradation rate of FB₁ was calculated using the following formula: (1—concentration of FB₁ residual/concentration of FB₁ original) × 100%.