A scientific relational database combined with a report generator for endoscopy in networks: EndoNet.

BACKGROUND AND STUDY AIMS: The flexibility required in academic endoscopy units is not provided by the available database systems. In a project involving substantial cooperation between endoscopists and computer scientists, we have developed an adaptable database, combined with a report generator embedded in the hospital's intranet. PATIENTS AND METHODS: Six workstations in different areas of the hospital were clustered with a UNIX operating system to implement multi-user capability and access control. A relational database was used to design an application appropriate to the specific needs of the endoscopy unit in a teaching hospital engaged in scientific research. Both the terminology used in standardized endoscopy nomenclature and a free text block facility were included. A graphical user interface was developed to assemble pertinent data, generate the reports, and supervise the database. RESULTS: A total of 4936 examinations including 2988 patients were entered consecutively during continuous routine operation of the system. Complete report generation required five minutes (median; range 1-9 minutes). Both structured items and free text were used in all the reports. Querying of the database was possible, concerning matters such as the need for repeated endoscopic therapy in acute gastrointestinal bleeding (4%), the search for Helicobacter pylori in appropriate patients (64%), the rate of accidental pancreatic duct visualization in endoscopic retrograde cholangiography (24%), and links between examinations and active trials (2%). Indicating improved report quality, the number and the diameter of esophageal varices in patients with varices were more frequently reported with the new report system than with previous typed reports (P<0.001). An anonymous questionnaire revealed that the readability of the computer-generated reports was better than that of the previous typewritten reports (P=0.01). CONCLUSIONS: This report describes the creation of a database application and a report generator meeting the needs of scientific and routine use, and the successful application of this system in an academic endoscopy unit.

Acute effect of ursodeoxycholic acid on gallbladder volume in healthy subjects.

BACKGROUND: Although it has been shown that chronic administration of ursodeoxycholic acid increases gallbladder fasting and residual volume, it is unknown whether ursodeoxycholic acid exerts an acute effect on gallbladder volume. We therefore evaluated the effect of a single oral dose of ursodeoxycholic acid on gallbladder volume in healthy volunteers. METHODS: After the volunteers had fasted overnight, gallbladder volume was measured sonographically every 15 min for 5 h. Following a 1-h control period group I (n = 8) received ursodeoxycholic acid (1000 mg) orally with 100 ml of water, whereas group II (n = 8) received 100 ml of water (placebo) only. Gallbladder volumes were calculated, applying the sum-of-cylinders method. Serum levels of ursodeoxycholic acid were determined by gas chromatography at 1-h intervals. RESULTS: Gallbladder fasting volumes before ursodeoxycholic acid were similar in both groups (24.0 +/- 2.3 ml versus 25.4 +/- 3.3 ml; NS). After ingestion of ursodeoxycholic acid (group I) gallbladder volume increased rapidly, reaching 27.6 +/- 3.1 ml (p < 0.04) 1 h and 38.4 +/- 3.4 ml (p < 0.02) 4 h after ingestion of ursodeoxycholic acid. The individual gallbladder volumes after ingestion of ursodeoxycholic acid in group I increased to 146%-211% of pretreatment values. Ursodeoxycholic acid serum levels increased from 0.94 +/- 0.38 mumol/l to 10.51 +/- 1.36 mumol/l (p < 0.001) and correlated closely with gallbladder volumes (r = 0.80; p < 0.05). After ingestion of water only (group II) gallbladder volume decreased transiently from 15 min to 30 min after water intake and then remained at pretreatment values throughout the study period. CONCLUSION: Administration of a single oral dose of ursodeoxycholic acid causes a rapid increase in gallbladder volume, which reaches 163 +/- 10% of pretreatment volume at 4 h and is closely correlated with ursodeoxycholic acid serum levels.

Simvastatin added to ursodeoxycholic acid does not enhance disappearance of gallstone fragments after shock wave therapy.

Inhibitors of the HMG-CoA reductase have been shown to further reduce the biliary cholesterol saturation in patients treated with oral bile acids for cholesterol gallbladder stones. It was the aim of our study to evaluate the efficacy of simvastatin in addition to ursodeoxycholic acid in the dissolution of gallstone fragments after shock wave lithotripsy and adjuvant bile acid dissolution therapy. Eighteen patients with a single radiolucent gallbladder stone and a serum cholesterol of more than 250 mg/dl were randomly assigned to receive either ursodeoxycholic acid alone (750 mg per day, group A, n = 9) or in combination with simvastatin (20 mg per day, group B, n = 9) for the dissolution of the gallstone fragments generated by extracorporeal shock wave lithotripsy. The two groups were well matched regarding their baseline characteristics. At the primary end point of the study 6 months after lithotripsy, there was no difference between the groups in the rate of gallstone disappearance with 4 of 9 patients being stone free in each group. As evaluated by life table analysis, even further follow-up showed no significant difference between the groups (P = 0.8). In group B, serum cholesterol levels decreased by 22% at 3 months (P = 0.01 vs. baseline) and by 24% at six months (P = 0.02) during treatment while no significant change was observed in group A. With both regiments, no adverse effects were observed. While simvastatin added to ursodeoxycholic acid resulted in a decrease of elevated serum cholesterol levels in gallstone patients, it did not enhance stone disappearance after shock wave lithotripsy and adjuvant bile acid dissolution therapy.

Agonist-regulated phosphorylation of the pancreatic cholecystokinin receptor.

The present study was undertaken to determine if the cholecystokinin (CCK) receptor may be phosphorylated, and to gain insight into its regulation. For this, the ATP pool of rat pancreatic acini was prelabeled with 32P, and the cells were stimulated with various secretagogues. CCK receptors from treated cells were enriched by sequential fractionation to produce plasmalemma, and subsequent solubilization and lectin-affinity chromatography. This protocol detected a phosphorylated Mr = 85,000-95,000 plasma membrane glycoprotein with features similar to the CCK receptor. Phosphorylation of this protein occurred rapidly (less than 2 min) and in a concentration-dependent manner in response to CCK, and was inhibited by the CCK receptor antagonist L-364,718. Further evidence that this represented the CCK receptor included comigration of phosphorylated and CCK radioligand affinity-labeled proteins on sodium dodecyl sulfate-polyacrylamide gels, both in native forms and after endoglycosidase F deglycosylation, and the specific adsorption of the phosphoprotein to a CCK analogue affinity resin. Phosphorylation occurred predominantly on serine residues of the receptor protein. Phosphorylation of this protein was also enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in response to direct activation of this enzyme by 12-O-tetradecanoylphorbol 13-acetate. Thus, the pancreatic CCK receptor is phosphorylated in a regulated manner, in response to both homologous and heterologous secretagogues, and to protein kinase C activation.

Use of photoaffinity probes containing poly(ethylene glycol) spacers for topographical mapping of the cholecystokinin receptor complex.

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of the cholecystokinin receptor on CHP212 human neuroblastoma cells.

Cholecystokinin (CCK) receptors reside on a large number of cell types along the digestive tract and in the nervous system. A human neuroblastoma cell line (CHP212) has recently been described to express a type A receptor, with structural specificity similar to that on pancreatic acinar cells and gall bladder smooth muscle cells but different from the predominant type of binding site found in brain (type B). In this work, we have performed photoaffinity labeling and protease peptide mapping of the CHP212 receptor and have compared it to other type A CCK receptors. 125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)-CCK-26-33], a probe that possesses a photolabile residue at position 33 within the theoretical receptor-binding domain of this hormone, specifically labeled a Mr = 80,000-90,000 glycoprotein on this cell line, while labeling larger proteins (Mr = 85,000-95,000) on rat pancreas and human gall bladder. Deglycosylation with endo-beta-N-acetylglucosaminidase F yielded bands of Mr = 43,000 from CHP212 and gall bladder and Mr = 42,000 from pancreas. Peptide mapping of the deglycosylated bands using Staphylococcus aureus V8 protease demonstrated identical patterns in CHP212 and gall bladder and a similar but different pattern in pancreas. Thus, although possessing heterogeneity in their carbohydrate domains, CCK receptors on human neuroblastoma cells (CHP212) and human gall bladder smooth muscle cells have highly similar or identical protein cores. The core protein on another type A CCK receptor, from rat pancreas, appears to differ from these, likely representing molecular heterogeneity between species.

The efficiency of covalent labeling of the pancreatic cholecystokinin receptor using a battery of crosslinkable and photolabile probes.

Affinity labeling is a powerful method for biochemical characterization of hormone receptors, dependent on approximation of reactive groups on ligand and receptor. In this work, we have compared the efficiency of covalent labeling of the rat pancreatic cholecystokinin (CCK) receptor by decapeptide probes with differing photolabile moieties sited at their amino-terminus, mid-region, or carboxyl-terminus, or chemically crosslinkable via their amino-terminus. Each labeled the same M(r) = 85,000-95,000 plasma membrane glycoprotein with a protein core of M(r) = 42,000. Affinity labeling this band through the amino-terminus of the decapeptide, 125I-D-Tyr-Gly [(Nle28,31)CCK-26-33], was inefficient using bifunctional chemical reagents, m-maleimidobenzoyl-N-hydroxy-succinimide ester (0.07% of total incubated radioactivity, representing 0.4% of specifically bound counts) or disuccinimidyl suberate (0.02, 0.2%) or a photolabile carbene precursor (0.06, 0.2%). A benzophenone at this locus yielded more efficient labeling of this band (0.09, 11.8%), but high levels of nonspecific labeling. Probes attached through residues within the receptor-binding domain were particularly useful. Photolabile derivatives of phenylalanine at the carboxyl-terminus of this domain yielded better incorporation (4-nitro-Phe33: 0.23, 1.4%; 4-azido-Phe3: 0.67, 6.0%). A 6-nitro-Trp30 derivative in the middle of this domain gave similarly efficient labeling (0.08, 3.5%) despite being a less potent pancreatic secretagogue. These studies clearly demonstrate that the efficiency of covalent labeling of a receptor can be markedly affected by the nature and site of crosslinking chosen.

Protease peptide mapping of affinity-labeled rat pancreatic cholecystokinin-binding proteins.

Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor.

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel tool for the study of cholecystokinin-stimulated pancreatic enzyme secretion.

The molecular events that mediate cholecystokinin (CCK)-stimulated pancreatic secretion are not well defined because of the complex receptor-binding and concentration-response characteristics of this hormone. Functional models of receptor occupancy initiating the cascade leading to secretion have been complicated by the inhibition of secretion effected by supramaximal concentrations of CCK. Recent report of a CCK analogue that does not exhibit supramaximal inhibition led us to synthesize a similar analogue that could also be radiolabeled for studies of receptor binding and affinity labeling, and for studies of second messenger activity. This probe, D-Tyr-Gly-[(Nle28,31)CCK-26-32]-phenethyl ester, was a fully efficacious secretagogue with no supramaximal inhibition, and, unlike native hormone, bound to a single class of sites present on both acini and membranes. Occupation of this site correlated well with stimulation of secretion. Evidence that this was indeed a CCK-binding site were the abilities of CCK and the antagonist L-364, 718 to inhibit binding of this analogue. Affinity labeling confirmed the identity of the site mediating secretory stimulation as a Mr = 85,000-95,000 protein. Whereas the nonhydrolyzable guanosine triphosphate analogue, 5'-guanylyl-imidodiphosphate, was a potent inhibitor of CCK binding, it had no effect on binding of this secretagogue, suggesting that a novel cascade not involving a guanine nucleotide-binding protein mediates CCK stimulation of pancreatic secretion.