Protease activity in zebrafish water samples.

(A) Michaelis-Menten plot with velocity as a function of [S-2238]. Each sample was maintained for 30 min prior to mixing with substrate under the specified conditions. Standard error bars (±0.07) were removed to improve clarity of each graph. (B) The proteins secreted by zebrafish were separated on a 5–20% Tris-glycine SDS-polyacrylamide gradient gel. The samples loaded in the lanes are as follows: 1, molecular weight marker (numbers on the left side indicate the size of the band in kDa); 2, 3, ZW; 4, zebrafish mouth wash; 5, zebrafish nose wash; and 6, zebrafish gill wash. Lanes 1 and 2 were stained with Coomassie blue dye. Other lanes were stained using 10 mM S-2238 following renaturation. Arrows indicate protease activity. (C) Western blot analysis of ZW. Arrows indicate trypsins. The numbers on the left side indicate the size of the molecular weight markers in kDa.

Immunohistochemistry of trypsin in zebrafish.

Zebrafish were dissected and sections of mouth, nose, gill, and pancreas were stained with H&E (1) and subjected to immunohistochemistry analysis using polyclonal rabbit antisera against human trypsin (PRAHT) (2). Alexa goat anti-rabbit IgG 488 antisera were used as the secondary antibody. Non-immunized rabbit IgG was used in control experiments (images not shown). 20X lens was used.

Detection of trypsin by confocal microscopy.

Confocal DIC images (a, 60X and b, 100X) of zebrafish sections of mouth, nose, gill and pancreas stained by immunohistochemistry using primary antibody (a) PRAHT, (b) Non-immunized rabbit IgG. Alexa Fluor 488 goat anti-rabbit IgG was used as secondary antibody. We used two channel image acquisition to examine the expression of trypsins that were probed with Alexa Fluor. A 488 nm and a 405 nm laser were used to excite the Alexa Fluor (Green) and autofluorescence (Blue), respectively. Columns 1 and 2 show brightfield and fluorescence images respectively.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Effects of ZW on gill bleeding, thrombocyte activation, and PAR-induced signaling.

(A) The zebrafish were subjected to the gill bleeding assay as described in Materials and Methods with the following conditions: 1, distilled water (DW); 2, 70 ng ZW; 3, 70 ng of ZW with 140 ng of PRAHT; and 4, 70 ng of ZW with 140 ng of control IgG. (B) Plate tilt assay measuring thrombocyte function: 1, PBS; 2, 7 ng ZW; 3, 7 ng ZW with 14 ng control IgG;and 4, 7 ng ZW with 14 ng PRAHT. Note the aggregated blood in 2 and 3 is more firm than the control blood 1 and blood 4. (C) Measurement of the functional activity of ZW on thrombocytes by annexin V binding. 7 ng of ZW, 14 ng of PRAHT and 14 ng of control IgG were used. The intensity of images from FITC-annexin fluorescence (green) was measured as mean pixels using Adobe Photoshop software version 7.0. (T test, n = 12). (D) ZW-induced ⁴⁵Ca²⁺ release from Xenopus oocytes microinjected with PARs mRNA. The data is presented as the mean value and standard error for 12 oocytes for each PAR mRNA.

Effect of PAR tethering peptides on thrombocyte function.

Top panel: Effect of PAR tethering peptides on thrombocyte aggregation. Plate tilt assay measuring thrombocyte function. (A) DAEFRH (control peptide). (B) TDEQTE (PAR1-21D). (C) SFSGFF (PAR1-5A). (D) PTQQTL (PAR1-21A). (E) SLVVIQ (PAR2-21A). (F) GFTGEE (PAR2-21D). Note the aggregated blood in (C), (D) and (E) is firmer than the control blood (A) and blood (B) and (F). 5 ng of each peptide was used. Middle panel: Measurement of functional activity of PAR tethering peptides on thrombocytes by annexin V binding. 5 ng of each tethering peptide was used. The intensity of images from FITC-annexin fluorescence (green) was measured as mean pixels using Adobe Photoshop software version 7.0 (T test, n = 12). Bottom panel: Effect of SLVVIQ tethering peptides on gill bleeding. The zebrafish were subjected to the gill bleeding assay as described in Materials and Methods with the following conditions: (A) 50 ng of DAEFRH (control peptide) and (B) 50 ng of SLVVIQ (PAR2-21A).

RT-PCR of trypsins in gill, mouth, and nose from zebrafish. Tissues from each organ were obtained using microscissors. RNA from tissues was isolated using Absolutely RNA prep kit from Stratagene, Inc. Trypsin primers were designed using MALDI-TOF data and NCBI database (Forward 5′-TCATGCTGATCAAGCTGA-3′ Reverse 5′-ATCCAGCGCAGAACATG-3′).

Effect of trypsin on gill bleeding. The zebrafish were subjected to the gill bleeding assay as described in the Methods with the following conditions: (A) Distilled water (DW), (B) 200 ng bovine trypsin, (C) 100 ng of bovine trypsin with 200 ng of PRAHT, and (D) 200 ng of bovine trypsin with 400 ng of control IgG.

The purified trypsins were separated on a 5–20% Tris-glycine SDS-polyacrylamide gradient gel. The values in the bottom panel represent the S-2238 cleaving activity of 1 ng of each sample at A_405nm and the mean of three independent determinations ± the standard error.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.