1. Volume 131, Issue 5, Pages 1463-1474 (November 2006)
Production, Secretion, and Biological Activity of the C-Terminal Flanking Peptide of Human Progastrin 
Kelly A. Smith, Oneel Patel, Shamilah Lachal, Ian Jennings, Bruce Kemp, John Burgess, Graham S. Baldwin, Arthur Shulkes 
Gastroenterology 
Volume 131, Issue 5, Pages (November 2006)
DOI: /j.gastro
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2. Figure 1
Structure of human progastrin and progastrin-derived peptides. Preprogastrin (101 amino acids) is converted to progastrin (80 amino acids) by removal of the signal peptide. The sequential actions of prohormone convertases converts progastrin to glycine-extended forms and the C-terminal flanking peptide (CTFP, shown by hatched box) by cleavage at paired basic residues. The C-terminus of Ggly is then amidated by peptidyl α-amidating monooxygenase. For simplicity, only the 17 amino acid forms of Ggly and Gamide are depicted.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Purification of progastrin-derived peptides from human antral tissue extracts by reverse phase HPLC. (A) Two nanomoles synthetic CTFP standard. (B and C) Human antral tissue extract from 2 different individuals. CTFP immunoreactivity (measured by RIA with antibody C14) is depicted by shaded bars, intact progastrin immunoreactivity (antibody 1137) is depicted by the dotted line, and the ACN gradient is depicted by the dashed line. A single unretained major peak of CTFP immunoreactivity was found in the column flow through in A and C. In B, a late eluting peak was observed in addition to the unretained peak.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Purification of CTFP by normal phase chromatography. (A) Synthetic CTFP standard and (B) unretained peak from the reverse phase HPLC from antral extract. The solid line depicts absorbance at 214 nm, and the dashed line depicts the ACN gradient.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Plasma displaced label from the anti-CTFP antibody in parallel with a CTFP standard. A CTFP standard curve (solid circles and solid line) was compared with dose response curves of plasma (open diamonds and dashed line) and extracted plasma (shaded triangles and dotted line) from a control subject. The standard curve is expressed as fmol/tube vs B/F, and the dose response curves are expressed as volume of plasma (μL) vs B/F. Dot points indicate individual data points, and lines represent curves of best fit for each data set generated by the program Sigmaplot as described in the Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Purification of CTFP by sizing chromatography. (A) Elution profile of 2 pmoles CTFP, 1 pmole progastrin, and 1 pmole Gamide standards detected by RIA with antibodies C14 (CTFP, progastrin) and 1296 (Gamide). (B) Human antral tissue extract. (C) One milliliter human plasma from a control subject. (D) One milliliter ethanol extracted plasma from a control subject. The elution profile of CTFP immunoreactivity is represented by solid circles and solid line and Gamide immunoreactivity by open circles and dotted line. CTFP immunoreactivity is detected in the early eluting material in both the standard and the plasma profiles but is not present in the extracted plasma. Plasma CTFP immunoreactivity elutes in a broad peak starting at the elution position of the synthetic standard.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
The concentration of CTFP and Gamide in the plasma of individuals following meal stimulation after an overnight fast. (A) The concentration of CTFP (solid circles and solid line) and (B) Gamide (open circles and dotted line) in pmol/l. Expressed as mean ± SEM, where n = 4 for Gamide and 7 for CTFP. The maximum concentration of CTFP is observed at 120 minutes after feeding, whereas Gamide concentrations reach a maximum earlier at 30 minutes after feeding.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
(A) The concentration (mean ± SEM) of Gamide, Ggly, progastrin, and CTFP in the plasma of normogastrinemic (solid bars, n = 14) and hypergastrinemic (shaded bars, n = 28) MEN-1 patients. Although Gamide was significantly higher in the hypergastrinemic compared with normogastrinemic patients (*P < .001), no difference was observed in the plasma concentration of CTFP and Ggly between groups. (B) The concentration (mean ± SEM) of Gamide, Ggly, and CTFP in the plasma of control subjects (solid bars, n = 42) and pernicious anemia patients (shaded bars, n = 26). Peptide concentrations (in pmol/L) were determined by RIA and are expressed as mean ± SEM. Gamide and Ggly were significantly higher in pernicious anemia patients compared with controls (*P < .001); however, no difference was observed in the concentration of CTFP.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
(A) CTFP immunostaining in a representative section of a human CRC (original magnification, ×200). (B) Control section in which nonimmune rabbit serum was used instead of the primary CTFP antibody.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
CTFP stimulates proliferation in gastric and colorectal carcinoma cell lines. Proliferation of (A) LIM1839, (B) SW1222, and (C) HCT-15 cells was measured by tritiated thymidine incorporation after 24, 48, and 24 hours incubation, respectively, with increasing concentrations of CTFP (100 pmol/L to 1 μmol/L; solid bars) and a single concentration of Ggly (10 nmol/L; hatched shaded bar) and compared with control (solid bar). Proliferation was measured in counts (cpm) and is expressed as a percentage of control. Percentages are depicted as mean ± SEM, where n = 3 experiments (6 replicates per experiment). Statistical significance, compared with control, was determined by a 1-way analysis of variance, followed by a Bonferroni t test, where +P < .01 and *P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
CTFP stimulates proliferation and migration of IMGE-5 cells. (A) Proliferation was measured by tritiated thymidine incorporation after 24 hours incubation with 10 nmol/L Ggly or CTFP and is expressed as a percentage of control. Percentages are depicted as mean ± SEM, where n = 5 experiments (4 replicates per experiment). (B) Images of wounded IMGE-5 monolayers in a migration assay at 0, 17, and 24 hours of treatment with 10 nmol/L Ggly or CTFP. (C) Graphical representation of wound healing assay on IMGE-5 cells. Cellular migration was determined by the extent of wound closure. Wound size at 17 and 24 hours is expressed as percentage of original wound size at t = 0, and the value at each time point is compared with the corresponding control at the same time point. Percentages are depicted as mean ± SEM, where n = 3 experiments (6 replicates per experiment). Statistical significance, compared with control, was determined by a 1-way analysis of variance, followed by a Bonferroni t test, where #P < .05, and *P < P < .05 compared with 24-hour treatment with 10 nmol/L Ggly.
Gastroenterology  , DOI: ( /j.gastro )
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12. Figure 11
CTFP stimulates MAPK phosphorylation in HCT-15 cells. (A) Western detection of 42- and 44-kilodalton forms of MAPK (p42 and p44, respectively) probed for phospho-MAPK (top panel) and total MAPK (bottom panel). Cells were treated with 10 nmol/L CTFP for 0, 3, 5, 10, 15, and 30 minutes. (B) Graphical representation of data from 3 independent experiments, mean ± SEM. The percentage of MAPK phosphorylation was determined as the ratio of optical density of the phosphorylated MAPK band compared with the total MAPK band. MAPK phosphorylation was significantly stimulated in HCT-15 cells after 3 and 5 minutes of treatment with 10 nmol/L CTFP (#P < .05).
Gastroenterology  , DOI: ( /j.gastro )
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13. Figure 12
Comparison of effects of progastrin6–80, G17-CTFP, and CTFP on MAPK phosphorylation in HCT-15 cells. (A) Western detection of 42- and 44-kilodalton forms of MAPK (p42 and p44, respectively) probed for phospho-MAPK (top panel) and total MAPK (bottom panel). Cells were treated with the indicated peptides with and without the CCK-2 antagonist for 3 minutes. (B) Graphical representation of data from 3 independent experiments, mean ± SEM. The percentage of MAPK phosphorylation was determined as the ratio of intensity of the phosphorylated MAPK band compared with the total MAPK band. MAPK phosphorylation in HCT-15 cells was significantly stimulated by all peptides. The CCK-2 antagonist had no effect alone and did not alter the stimulatory effect of CTFP (#P < .05).
Gastroenterology  , DOI: ( /j.gastro )
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