Fig 2: Kinetics of cysteine protease activities in diabetic (DM) and normal (NL) rat livers. Tissue extracts (2-6 µL containing 50 µg protein) were preactivated by incubation of tissue extract aliquots with 2 mM dithiothreitol (10 min, room temperature). The following substrates were then added (20 µM, 200 µL final volume): Z-FR-MCA (substrate for cysteine proteases), or e-NH2-caproyl-Cys(Bzl)-Cys(Bzl)-MCA (substrate for cathepsin B). The fluorescence produced upon hydrolysis of the substrates was measured every 20 s in FlexStation 3 (Molecular Devices, USA), using ?exc=380 nm and ?emi=460 nm. The assays were also performed in the presence of inhibitors: 1 mM phenylmethylsulfonyl fluoride (PMSF, inhibitor of serine-proteases), 5 µM E64 (irreversible inhibitor of cysteine-proteases), and 1 µM CA074 (irreversible inhibitor of cathepsin B). A maximum of 10% substrate consumption was considered, and each point represents the mean±95% confidence interval of four replicates for all animals in each group.

Fig 3: (A) Detection of glucose-regulated protein 78 (GRP78) autoantibodies in the patient’s serum IgGs by a Western blot analysis. Human full-length GRP78 recombinant protein (1 µg, Abcam, Cambridge, UK) as the antigen was fractionated and transferred to a polyvinylidene difluoride (PVDF) membrane. IgG isolated (5 µg/mL) from this patient before (NMO-T) and after (NMO+T) treatment, NMO-IgG (positive control: PC), healthy control (HC)-IgG, and anti-GRP78 antibodies (dilution 1: 200; Abcam) were used as primary antibodies, and anti-human secondary HRP-conjugated antibody (dilution 1: 4,000) was used as the secondary antibody. Bands corresponding to the variant form of 60-kDa GRP78 (red arrowhead) were observed in IgGs from NMO-T, NMO+T and PC samples but not from HCs. Bands corresponding to 80-kDa GRP78 (black arrowhead) were observed in IgGs from the PC sample. The bands were visualized by chemiluminescence (ImmunoStar® LD; Wako, Osaka, Japan). (B) Permeability coefficient of 10-kDa dextran across human brain endothelial cell lines. Adult human brain microvascular endothelial cells were cultured in 24-well collagen-coated Transwell tissue culture inserts (0.4-mm pore size; Corning, Corning, USA). After exposure to IgG from the patient before (NMO-T) and after (NMO+T) treatment and from two healthy controls (HC1 and HC2), FITC-dextran fluorescence (10 kDa, final concentration 1 mg/mL; Sigma-Aldrich, St. Louis, USA) was added to the luminal insert, and 100 µL of medium was collected from the abluminal chamber over 40 minutes. Fluorescence signals were measured at a wavelength of 490/520 nm (absorption/emission) using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices, San Jose, USA). Both NMO-T and NMO+T-IgGs significantly increased the 10-kDa dextran permeability compared to HC1 and HC2 (*p<0.05, **p<0.01).

Fig 4: Convergent compounds identified through ISCA of Kampo and TCM formulations for pain. A. Summary of convergent compounds represented in both Kampo and TCM formulations. These include terpenes (orange sector) and alkaloids (blue sector), with specific compounds illustrated. B. Terpenes found in the ISCA include effective ligands and potential agonists-desensitizers for nociceptive TRP channels. HEK cells inducibly expressing the indicated ion channels were loaded with Fluo-4 acetoxymethyl ester in a modified Ringer solution containing 1 mM CaCl2 Cells were stimulated with vehicle or the indicated terpene at a concentration of 1 µM, or matched vehicle, and time-resolved fluorescence measurements were collected in a Molecular Devices Flexstation 3. The peak attained increases in relative fluorescence units (RFU) were calculated, vehicle subtracted and plotted. Comparison plots show the relative intensity of the intracellular free calcium mobilization initiated by each terpene (with the diameter of each circle representing the peak intensity, upper panel, and as histograms, lower panel). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)