Fapgg

Skipjack tuna roes were provided by Ningbo Today Food Co., Ltd. (Zhejiang, China). HUVECs were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). ROS (Product no. E004-1-1), Hoechst33342 (Product no. G023-1-1), CAT (Product no. A007-1-1), SOD (Product no. A001-3-2), GSH-PX (Product no. A005-1-2), MDA (Product no. A003-1-2), ET-1 (Product no. H093-1-2), and NO (Product no. A013-2-1) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Glutathione (GSH), trypsin, pepsin, papain, ACE and FAPGG were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (China). Alcalase, MTT, DMEM, DMSO, fetal bovine serum (FBS), norepinephrine (NE), trifluoro acetic acid (TFA), neutrase and Cap were purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). ACEi peptides of WGESF (TRP3), IKSW (TRP6), YSHM (TRP9), and WSPGF (TRP12) with purity higher than 95% were synthesized in Shanghai Apeptide Co. Ltd. (China).

ACE (1 Unit, rabbit lung), FAPGG (N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly), and captopril were purchased from Sigma Chemical Company (St Louis, MO, USA). All chemicals and solvents used for column chromatography were of reagent grade and purchased from commercial sources, unless otherwise stated.

Assessment of ACE activity was based on the spectrophotometric measurement of FAPGG hydrolysis [28] (link)
[29] (link). The reaction mixture (200 μL) contained 50 μL of serum, 0.5 mM FAPGG (N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine) (Sigma, St. Louis, MO, USA) substrate, 300 mM sodium chloride, and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 8.2. Measurement of ACE activity is based on the change in the absorption at 340 nm when FAPGG hydrolyzed to furylacryloyl-L-phenylalaline (FAP) and glycylglycine (GG). The reaction was performed in 96-well plates (Greiner Bio-One, Frickenhauser, Germany). Changes in FAPGG absorbance were detected using a microplate reader (NOVOstar; BMG Labtech GmbH, Offenburg, Germany). Hydrolysis of FAPGG by ACE was recorded in every 5 minutes at 37C°. Optical density values were plotted as a function of reaction time and fitted by linear regression. The fit and the data were accepted when r>0.9. ACE activity was calculated by the equation:
ACE activity = −(S/k)*D,
where S is the rate of observed decrease in optical density (1/min), k is the change in optical density upon the complete cleavage of 1 nmol of FAPGG, and D is the dilution of the serum. One unit (U) of ACE activity represents 1 nmol of FAPGG hydrolysis per minute at 37 °C.

TFA (trifluoroacetic acid), acetonitrile, trypsin (from bovine pancreas), papain (from pawpaw sap), FAPGG (N-(3-[2-furylacryloyl-Phe-Gly-Gly])), ACE (angiotensin-I converting enzyme) from rabbit lung and captopril (≥98% purity), were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). Ultrafiltration membranes with a 3 kDa cut-off were purchased from Millipore (Bedford, MA, USA). Analytical and semi-preparative columns were purchased from Macherey Nagel GmbH Co. (St. Neumann Neander, Düren, Germany). All other chemicals used were of analytical grades.

Skipjack tuna (K. pelamis) dark muscles were provided by Ningbo Today Food Co., Ltd. (China). Sephadex G-25 resins and nitric oxide (NO) assay kits (Nitrate reductase approach, A012-1) were purchased from Nanjing Jiancheng Bioengineering Institute (China). ET-1 ELISA kit (HM10108) was purchased from Shanghai Qiaodu Biotechnology Co., Ltd. (China). Trypsin, pepsin, papain, ACE, and FAPGG were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (China). Alcalase, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco's modified eagle medium (DMEM), penicillin-streptomycin liquid, dimethylsulfoxide (DMSO), fetal bovine serum (FBS), norepinephrine (NE), Neutrase and Cap were purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). ACEi peptides of STAP1-STAP14 with purity higher than 95% were synthesized in Shanghai Apeptide Co., Ltd. (China).

The levels of serum ACE and lysozyme were evaluated.
Serum ACE activity was measured using FAPGG (N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycylglycine; Sigma-Aldrich) with a Buhlmann kit [12 (link)]. Normal values for adults are 20–70 U/I and 29–112 U/I for children under 18 years old.
Serum lysozyme activity was measured by using radial immunodiffusion (Mancini G. et al.) [13 (link)], with NANORID™ kits (Binding Site Ltd., Birmingham, UK). Normal values are 9.6–17.1 mg/L for all ages.
The means of serum ACE and lysozyme values in the ocular sarcoidosis groups (proven and suspected) were compared to a control group of non-granulomatous uveitis (pars-planitis and HLA B27 related uveitis) for which these tests had been performed as part of the work-up before the final diagnosis was known. The control groups were chosen based on the availability of each test.
Furthermore, patients were classified into four groups regarding elevation of both tests to compare their proportion: (1) both tests normal; (2) ACE elevated, lysozyme normal; (3) ACE normal, lysozyme elevated; and (4) both tests elevated.