Elastase from porcine pancreas

Folin–Ciocalteu’s phenol reagent, aluminum chloride (AlCl₃), dimethyl sulfoxide (DMSO), sodium acetate (NaOAc), quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), L-ascorbic acid, potassium persulfate (K₂S₂O₈), elastase from porcine pancreas, epigallocatechin gallate (EGCG), N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), tyrosinase from mushroom, kojic acid (KA), and 3,4-dihydroxy-L-phenylalanine (L-DOPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate (Na₂CO₃) was purchased from Merck (Darmstadt, Germany). Gallic acid was purchased from TCI America (Portland, OR, USA). Dipotassium phosphate (K₂HPO₄) and monobasic potassium phosphate (KH₂PO₄) were purchased from HiMedia (Mumbai, India). Tris base was purchased from Vivantis Technologies (Shah Alam, Malaysia). Ethanol and methanol were purchased from RCI Labscan (Bangkok, Thailand). All chemicals and reagents were analytical grades.

HEK293 cells and HeLa cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum and antibiotics. Cells were transfected with plasmid DNAs encoding TRPC3, TRPC6, or TRPC7 by using X-tremeGENE™ 9 reagent (Roche, Basel, Switzerland) according to manufacturer’s instruction. Mouse aortic smooth muscle cells were isolated from aorta of 6–8 weeks old 129/Sv mice. Thoracic aorta was removed and cleaned from perivascular fat. The aorta was incubated with enzyme solution (1 mg/mL collagenase type 2 (Worthington, Columbus, OH, USA), 1 mg/mL soybean trypsin inhibitor (Sigma, St. Louis, MO, USA), and 0.75 units/mL elastase from porcine pancreas (Sigma, St. Louis, MO, USA) dissolved in Hank’s balanced salt solution (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 37 °C. Adventitia and endothelium were then removed. Remaining medial layer was cut into small pieces and incubated with enzyme solution for 1–1.5 h at 37 °C. After trituration, cells were plated and cultured in DMEM supplemented with 20% FBS and penicillin/streptomycin at 37 °C with 5% CO₂. Cells were used between 3rd to 7th passages.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Thermo Scientific, (Waltham, MA, USA). The phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-EDTA were obtained from Gibco (Gaithersburg, MD, USA). Sodium carbonate (Na₂CO₃), sodium hydrogen carbonate (NaHCO₃), disodium hydrogen phosphate (Na₂HPO₄), potassium dihydrogen phosphate (KH2HPO4), and sodium chloride (NaCl) were obtained from Ajax Finechem Pty Limited (Taren Point, Australia). Polyvinyl alcohol (PVA) was obtained from Chem-Supply Pty Ltd. (Gillman, Australia). Direct red 80, picric acid, human collagen type I, ascorbic acid, L-DOPA, α-MSH, tyrosinase enzyme (≥2000 units/mg solid), collagenase from Clostridium histolyticum (0.25–1.0 FALGPA units/mg solid, ≥125 CDU/mg solid), elastase from porcine pancreas (lyophilized powder ≥ 50 units/mg protein), and gelatin type B were obtained from Sigma-Aldrich (St. Louis, MO, USA). B16F10 (murine melanoma cell line), human keratinocyte cell line (HaCaT), and primary normal human dermal fibroblasts (NHDF) were purchased from American Type Culture Collection, Manassas, VA, USA.

All crystals were grown at 20 °C using either the sitting or hanging drop vaporization methods. For RC3H1, the His6-tag-removed RC3H1 (a.a. 1–445) protein at a 25 mg/mL concentration was first mixed with elastase from porcine pancreas (Sigma-Aldrich) at a ratio of 1:1000 (w/w), then immediately mixed with a reservoir solution containing 25% PEG-8000, 0.2 M NaCl, 0.1 M HEPES, pH 7.5, 5% ethyl glycol at a ratio of 1:1. Crystals of the apo-form RC3H2 RNA-binding region were grown in a sitting drop mixed from 2 μL protein solution (C-terminal His6-tagged, 29 mg/mL) with 1 μL well solution containing 3.2 M NaCl, 0.1 M sodium acetate, pH 4.6. For the RC3H2/Ier3 RNA complex, N-terminal His6-tag-removed RC3H2 protein was first diluted to 6.7 mg/ml, then mixed with Ier3 RNA solution (2 mM) at a molar ratio of 1:1.5 before being set in a hanging drop. The drop was mixed from 1.5 μL protein/RNA complex and 1.5 μL well solution consisting of 17% PEG-10K, 0.1 M Bis-Tris, pH 6.5, and 5% ethyl glycol. All crystals for data collection were cryo-protected via an initial immersion in the corresponding well solution plus 10–15% ethylene glycol, then an immersion in Paratone-N (Hampton Research), before being flash-frozen in liquid nitrogen.

Iscove’s modified Dulbecco’s medium (IMDM), Dulbecco’s Modified Eagles medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). N-Succinyl-Ala-Ala-Ala-p-nitroanilide (STANA), elastase from porcine pancreas, all-trans retinol, and adenosine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Procollagen Type I C-peptide (PIP) EIA kit was purchased from Takara Bio Inc. (Kusatsu, Shiga, Japan). Human Total MMP-1 DuoSet ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA).

Inhibition of elastase was based on described corresponding assays [45 (link)], with following modifications: elastase from porcine pancreas (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.2 M buffer solution (Tris-HCl, Sigma-Aldrich, St. Louis, MO, USA). In addition, inhibitor (10 µL) or fraction dilution (10 µL), enzyme (10 µL) and buffer solution (150 µL) were added to the wells and the plate was preincubated for 15 min at 25 °C, after which substrate (30 µL, N-succinyl-Ala-Ala-Ala-p-nitroanilide, Sigma-Aldrich, St. Louis, MO, USA) was added and the plate was incubated at 25 °C. The absorbance was read at 405 nm after 10 min. Six concentrations (5, 12.5, 25, 37.5, 50, and 125 µg·mL⁻¹) of the inhibitor elastatinal (Sigma-Aldrich, St. Louis, MO, USA) in 1% DMSO were used as positive control.

Peanuts were grown on Buyeo 153 farm (Buyeo, Chungcheongnam-do, Korea). The kernels of the peanuts were removed and only the shells were recovered. Peanut shells (PSs) were washed with DW and dried at 110 °C. After that, PSs were prepared with a size of less than 90 μm using a test sieve and used as biomass. Ethanol (EtOH, 94.50%), methanol (MeOH, 99.9%), acetone (Ace, 99.7%) and ethyl acetate (EA, 99.9%) were purchased from Samchun Chemical (Gangnam, Seoul, Republic of Korea). Folin–Ciocalteu reagent, sodium carbonate (Na₂CO₃), luteolin, gallic acid, sodium nitrite (NaNO₂), 2,4,6-tripyridyl-S-triazine (TPTZ), iron (II) sulfate (FeSO₄), iron (III) chloride (FeCl₃), sodium acetate trihydrate (CH₃CO₂Na·3H₂O), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), N-Succinyl-Ala-Ala-Ala-p-nitroanilide and elastase from porcine pancreas were obtained from Sigma-Aldrich (St. Louis, MO, USA). Aluminum chloride (AlCl₃), calcium carbonate (CaCO₃), hexane (95%) and sulfuric acid (H₂SO₄) were purchased from Duksan Pure Chemical (Ansan-si, Gyeonggi-do, Republic of Korea). All reagents used in this study were analytical grade.