Benefits of semiology taught using near-peer tutoring are sustainable.

BACKGROUND: Near-peer tutoring appears to be an efficient approach for teaching clinical skills. However, the clinical experience gained in the form of student medical internships may offset any interest in such tutoring programme. We then investigated the long-term benefits of this programme. METHODS: This study was conducted in a medical school that experimented in near-peer tutoring for semiology intended for undergraduate medical students. Objective Structured Clinical Examinations and a written semiology test were used to assess students' clinical skills immediately on its conclusion and repeated one and 2 years after the tutoring was completed. RESULTS: 116 students were evaluated initially (80 tutored and 36 untutored), 38 at 1 year (16 tutored and 22 untutored), 42 at 2 years (21 tutored and 21 untutored). In the global score for Objective Structured Clinical Examinations: at 1 year, the tutored group scored 14.0 ± 1.05 and the untutored group scored 11.3 ± 2.3 (p < 0.001), at 2 years, the tutored group scored 15.1 ± 1.5 and the untutored group scored 12.4 ± 2.2 (p < 0.001). We found a similar but smaller difference for the written semiology test. The difference for Objective Structured Clinical Examinations between tutored and untutored students vanished over time for cross-cutting skills. CONCLUSIONS: Near-peer tutoring in semiology for undergraduate medical students led to better results that remained with the passing of time. Though internships do allow an improvement in the clinical skills of untutored students, they did not reach the level of tutored students.

Oral trivalent bismuth ions decrease, and trivalent indium or ruthenium ions increase, intestinal tumor burden in ApcΔ14/+ mice.

Immature forms of the peptide hormone gastrin have been implicated in the development of colorectal cancer (CRC). The biological activity of glycine-extended gastrin (Ggly) is dependent on the binding of Fe3+ ions in vitro and in vivo. The aim of the present study was to determine the effect of blocking Fe3+ ion binding to Ggly, using Bi3+, In3+ or Ru3+ ions, on the development of intestinal tumors in APCΔ14/+ mice. APCΔ14/+ mice were treated orally with Bi3+, In3+ or Ru3+ ions for up to 60 days, serum trace metals were analyzed by inductively coupled plasma mass spectrometry, and the incidence and size of intestinal tumors were assessed. Bi3+ treatment significantly decreased the number of tumors larger than 3 mm in male mice. In3+ or Ru3+ treatment significantly increased the tumor burden in all animals and In3+ increased the number of tumors larger than 3 mm or 5 mm in male mice alone. The fact that binding of In3+ or Ru3+ ions to Ggly was orders of magnitude stronger than the binding of Bi3+ ions implies that the inhibitory effect of Bi3+ ions is not a consequence of a reduction in Ggly activity. However, further testing of higher doses of Bi3+ ions for longer periods as an oral treatment for intestinal tumors is warranted.

Complexes of gastrin with In3+, Ru3+ or Ga3+ ions are not recognised by the cholecystokinin 2 receptor.

The peptide hormone gastrin (Gamide) binds trivalent metal ions, including indium (In), ruthenium (Ru) and gallium (Ga), with high affinity. Complexes of gastrin with chelated isotopes of In and Ga have previously been used for the location of tumours expressing the cholecystokinin 2 receptor (CCK2R). The aim of the present study was to purify the complexes of Gamide with radioactive isotopes of In, Ru or Ga and to investigate their ability to bind to the CCK2R. The radioactive Gamide complexes were purified on Sep-Pak C18 cartridges or by anion exchange HPLC. Binding to the CCK2R was assessed with a stably transfected clone of the gastric carcinoma cell line AGS. The 106Ru-Gamide complex could be eluted from the C18 cartridge; the 111In-Gamide and 68Ga-Gamide complexes bound irreversibly. All three complexes were successfully purified by anion exchange HPLC. The failure to detect binding of the 111In-Gamide, 106Ru-Gamide and 68Ga-Gamide complexes to the CCK2R suggests that formation of these complexes will not be useful for the detection of tumours expressing this receptor, but may instead provide alternative ways to block the actions of Gamide as a growth factor or a stimulant of gastric acid secretion. The complexes between the hormone gastrin and radioactive 111In, 106Ru or 68Ga ions were purified by anion exchange HPLC using a NaCl gradient. The failure to detect binding of the complexes to the cholecystokinin 2 receptor suggests that metal ion treatment may provide novel approaches to block the biological actions of gastrin.

Retro-inverso forms of gastrin5-12 are as biologically active as glycine-extended gastrin in vitro but not in vivo.

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function.

Activation by zinc of the human gastrin gene promoter in colon cancer cells in vitro and in vivo.

Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor ß-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis.

The C-terminal flanking peptide of progastrin induces gastric cell apoptosis and stimulates colonic cell division in vivo.

Progastrin (PG) is processed into a number of smaller peptides including amidated gastrin (Gamide), non-amidated glycine-extended gastrin (Ggly) and the C-terminal flanking peptide (CTFP). Several groups have reported that PG, Gamide and Ggly are biologically active in vitro and in vivo, and are involved in the development of gastrointestinal cancers. CTFP is bioactive in vitro but little is known of its effects in vivo. This study investigated the bioactivity of CTFP in vivo in normal tissues using gastrin deficient (GASKO) mice and in two mouse models of cancer (SCID mice bearing xenograft tumors expressing normal or knocked-down levels of gastrin and a mouse model of hepatic metastasis). As with Ggly, CTFP treatment stimulated colonic proliferation in GASKO mice compared to control. CTFP also significantly increased apoptosis in the gastric mucosa of male GASKO mice. CTFP did not appear to effect xenograft growth or the incidence of liver metastases. This is the first demonstration that CTFP has specific biological activity in vivo in the colon and stomach.

Identification of determinants of glucose-dependent insulinotropic polypeptide receptor that interact with N-terminal biologically active region of the natural ligand.

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.