Harvested white-tailed deer as sentinel hosts for early establishing Ixodes scapularis populations and risk from vector-borne zoonoses in southeastern Canada.

Due to recent establishment of the blacklegged tick, Ixodes scapularis Say, in southeastern Canada, tick-borne zoonoses (Lyme disease, human granulocytotropic anaplasmosis, and babesiosis) are of growing concern for public health. Using white-tailed deer (Odocoileus virginianus) culled in southwestern Quebec during 2007-2008, we investigated whether hunter-killed deer could act as sentinels for early establishing tick populations and for tick-borne pathogens. Accounting for environmental characteristics of culling sites, and age and sex of deer, we investigated whether their tick infestation levels could identify locations of known tick populations detected in active surveillance, presumed tick populations detected by passive surveillance, or both. We also used spatial cluster analyses to identify spatial patterns of tick infestation and occurrence of tick-borne zoonoses infection in ticks collected from the deer. Adult ticks were found on 15% of the 583 deer examined. Adult male deer had the greatest number (approximately 90%) of adult ticks. Overall, 3, 15, and 0% of the ticks collected were polymerase chain reaction (PCR)-positive for Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. Our statistical analyses suggest that sex and age of deer, temperature, precipitation, and an index of tick dispersion by migratory birds were significantly associated with tick infestation levels. Cluster analysis identified significant clusters of deer carrying ticks PCR-positive for A. phagocytophilum, and for deer carrying two or more I. scapularis. Our study suggests that hunter-killed deer may be effective as sentinels for emerging areas of tick-borne anaplasmosis. They may have limited use as sentinels for early emerging I. scapularis tick populations and emerging Lyme disease risk.

Associations between Ixodes scapularis ticks and small mammal hosts in a newly endemic zone in southeastern Canada: implications for Borrelia burgdorferi transmission.

Immature Ixodes scapularis infestation and Borrelia burgdorferi infection of wild small mammals were studied from June to October in 2007 and from May to October in 2008 at 71 study sites in a zone where I. scapularis populations and environmental Lyme disease risk are emerging in southwestern Quebec. Seasonal host-seeking activity of immature I. scapularis was similar to patterns reported previously in Canada and the USA: nymphal activity peaked in spring while larval activity peaked in late summer. Synchronous activity of nymphs with some larvae was observed in late spring, which could favour establishment of B. burgdorferi strains that cause short-lived infections in their hosts. White-footed mice (Peromyscus leucopus), deer mice (P. maniculatus), chipmunks (Tamias striatus), and red squirrels (Tamiasciurus hudsonicus) carried 92.0% of the larvae and 94.2% of the nymphs collected. Adult male white-footed mice carried significantly larger numbers of both larval and nymphal I. scapularis than other species and classes of small mammals (different demographic groups or physiological status: age, sex, sexual activity). We conclude that seasonality and host association were comparable to previous studies in North America, even in the context of a newly endemic pattern of low infection prevalence and low densities of host-seeking and feeding I. scapularis in southwestern Quebec. Our studies suggest that B. burgdorferi transmission cycles are focused on adult male mice (which carried 35% of all feeding ticks collected in the study), so control methods targeting this class of hosts may be particularly effective. However, our study also suggested that habitats containing a diverse host structure may dilute transmission cycles by partitioning of nymphal and larval ticks on different host species.

An ergonomic approach to reorganize parking inspection agents' work productivity, health and safety in São Paulo, Brazil.

OBJECTIVE: The Traffic Engineering Company of the City of São Paulo (Brazil) observed a decrease in productivity, and an increase in sick leave, accidents and psychological distress among their parking inspection agents. To document this situation, qualitative research was undertaken to obtain an in-depth comprehension of work activity. PARTICIPANTS: Workers, managers and health and safety professionals contributed to the documentation of the problem and to the proposal of possible solutions. METHODS: Ergonomic work analysis focusing on real work activity, as well as interviews with individual or groups of stakeholders, were conducted. RESULTS: This research revealed that political-economic factors gradually contributed to: 1) an increasing work load; 2) growing fatigue throughout the day, increasing the workers' vulnerability to incidents and accidents and their tendency to react inappropriately to violence experienced on the street; and 3) excessive individual responsibility to manage dangerous situations. CONCLUSIONS: Recommendations to ameliorate the situation are proposed. These suggestions are discussed in terms of feasibility given the impact of macro social factors upon micro work activity, and the associated potential expansion of the ergonomist's role.

Ixodes scapularis ticks collected by passive surveillance in Canada: analysis of geographic distribution and infection with Lyme borreliosis agent Borrelia burgdorferi.

Passive surveillance for the occurrence of the tick Ixodes scapularis Say (1821) and their infection with the Lyme borreliosis spirochaetes Borrelia burgdorferi s.l. has taken place in Canada since early 1990. Ticks have been submitted from members of the public, veterinarians, and medical practitioners to provincial, federal, and university laboratories for identification, and the data have been collated and B. burgdorferi detected at the National Microbiology Laboratory. The locations of collection of 2,319 submitted I. scapularis were mapped, and we investigated potential risk factors for I. scapularis occurrence (in Quebec as a case study) by using regression analysis and spatial statistics. Ticks were submitted from all provinces east of Alberta, most from areas where resident I. scapularis populations are unknown. Most were adult ticks and were collected in spring and autumn. In southern Québec, risk factors for tick occurrence were lower latitude and remote-sensed indices for land cover with woodland. B. burgdorferi infection, identified by conventional and molecular methods, was detected in 12.5% of 1,816 ticks, including 10.1% of the 256 ticks that were collected from humans and tested. Our study suggests that B. burgdorferi-infected I. scapularis can be found over a wide geographic range in Canada, although most may be adventitious ticks carried from endemic areas in the United States and Canada by migrating birds. The risk of Lyme borreliosis in Canada may therefore be mostly low but more geographically widespread than previously suspected.

Canadian environmental effects monitoring: experiences with pulp and paper and metal mining regulatory programs.

In Canada, Environmental Effects Monitoring (EEM) programs exist within two regulations: the Pulp and Paper Effluent Regulations and the new Metal Mining Effluent Regulations under the Canadian Fisheries Act. EEM provides a biological, effects-based feedback loop to assess the effectiveness of technology-based regulations in protecting receiving environments. The promulgation of the Pulp and Paper Effluent Regulations, in 1992, represented a significant step forward in the Canadian regulatory approach by incorporating directly into a regulation a requirement to assess the effects of effluent discharges on receiving environments using proven scientific monitoring methodologies. Similarly, an assessment of the aquatic impacts of mines resulted in recommendations to amend the Metal Mining Effluent Regulations, recently promulgated in 2002, and includes an EEM program as a science-based feedback loop. As such, these regulations recognize the possibility that national, technology-based standards may not necessarily protect all receiving environments because of the diversity and variability of both discharges and receiving sites across the country. Since that time, EEM has improved its flexibility by considering both advances in science and the uniqueness of monitoring sites across Canada to allow the most appropriate and cost-effective monitoring approaches at each site while maintaining national consistency. This paper discusses the use of monitoring under two Canadian regulations to assess effects on aquatic ecosystems. As well, the National EEM approach to maintaining up-to-date scientific practices in a national regulatory program is discussed using examples.

Two new peptides to improve post-operative gastric ileus in dog.

Peptides can influence gastrointestinal motility, and from data obtained earlier in rats, we hypothesized that MTL-RP/Ghrelin, as well as CGRP receptor antagonist 8-37, could improve gastric post-operative ileus in dog. Dogs submitted to laparotomy were perfused with or saline or CGRP 8-37 or MTL-RP/Ghrelin on days 1-4 post-operatively while gastric emptying was estimated by measuring the postprandial increase in plasma acetaminophen ingested with a meal. As expected, in saline-treated animals the gastric emptying function was impaired post-operatively. The total amount of acetaminophen emptied (AUC over 150 min) on post-operative days 1-4 reached respectively 31+/-5%, 65+/-8%, 60+/-8% and 62+/-8% of the normal emptying capacity. CGRP antagonist increased the total emptying of acetaminophen to 52+/-5% on day 1, 95+/-2% on day 2 and 103+/-3% (P<0.05) on day 3. The delayed emptying of acetaminophen seen post-operatively in saline-treated animals could be completely reversed by MTL-RP/Ghrelin (P<0.01) whether it was given at 100 microg/kg on day 2 (102+/-7% of the normal emptying capacity), 4 microg/kg on day 3 (106+/-7%) or 20 microg/kg on day 4 (132+/-8%). As found earlier in rodents, CGRP receptor antagonist 8-37 as well as MTL-RP/Ghrelin are potent prokinetics to improve post-operative gastric ileus in dog.

Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat.

A novel peptide called ghrelin or motilin-related-peptide (MTLRP) was found in the stomach of various mammals. We studied its effect on the motor function of the rat gastrointestinal tract. In normal, conscious unoperated animals, ghrelin/MTLRP (5 or 20 microg/kg iv) significantly accelerated the gastric emptying of a methylcellulose liquid solution (gastric residue after 15 min: 57 +/- 7, 42 +/- 11, 17 +/- 4, and 9 +/- 3% of the ingested meal with doses of 0, 1, 5, and 20 microg/kg iv, respectively) Transit of the methylcellulose liquid solution was also accelerated by ghrelin/MTLRP in the small intestine but not in the colon. Des-[Gln(14)]ghrelin, also found in the mammalian stomach, was as potent as ghrelin in emptying the stomach (gastric residue after 15 min: 12 +/- 3% at a dose of 20 microg/kg iv). In rats in which postoperative gastrointestinal ileus had been experimentally induced, ghrelin/MTLRP (20 microg/kg iv) reversed the delayed gastric evacuation (gastric residue after 15 min: 28 +/- 7% of the ingested meal vs. 82 +/- 9% with saline). In comparison, the gastric ileus was not modified by high doses of motilin (77 +/- 7%) or erythromycin (82 +/- 6%) and was only partially improved by calcitonin gene-related peptide (CGRP) 8-37 antagonist (59 +/- 7%). Ghrelin/MTLRP, therefore, accelerates the gastric emptying and small intestinal transit of a liquid meal and is a strong prokinetic agent capable of reversing the postoperative gastric ileus in rat.

Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Digestive motor effects and vascular actions of CGRP in dog are expressed by different receptor subtypes.

Calcitonin gene-related peptide (CGRP) is a 37 AA peptide localized in blood vessels and nerves of the GI tract. Activation of CGRP receptors (subtypes 1 or 2) usually induces vasodilation and/or muscle relaxation, but its effects in dog and on gastroduodenal motility are still unclear. This study looked for the effect of CGRP and the antagonist CGRP8-37, specific for CGRP type 1 receptor, 1) on GI motility (interdigestive and postprandial), and 2) on hemodynamy, in conscious dogs. During the interdigestive period, the infusion of CGRP1-37 (200 pmol/kg/h) or CGRP8-37 (2000 pmol/kg/h) did not modify the duration of the migrating motor complex nor the release nor the motor action of plasma motilin. The gastric emptying of a solid meal (15 g meat/kg) was reduced by the administration of CGRP1-37 (AUC: 2196 +/- 288.6 versus 3618 +/- 288.4 with saline or T12: 78 +/- 7.3 versus 50 +/- 4.3 min; P < 0.01) and this effect was reversed by the antagonist CGRP8-37. CGRP1-37 significantly (P < 0. 01) diminished arterial pressures (118 +/- 1.6/64 +/- 1.4 vs. 125 +/- 1.4/75 +/- 1.2 mmHg with saline) and accelerated the basal cardiac rhythm (110 +/- 1.4 versus 83 +/- 1.6 beats/min). However, CGRP8-37 failed to block the cardiovascular effects of CGRP1-37. In dog, CGRP could influence digestive motility by slowing the gastric emptying of a meal through an action on CGRP-1 receptors. Hemodynamic effects of CGRP were not blocked by CGRP8-37 and seem therefore mediated by CGRP-2 receptor subtype.

Neural and muscular receptors for motilin in the rabbit colon.

Motilin receptors were classically recognized in the gastroduodenal area, where they help to regulate interdigestive motility. More recently, motilin receptors were identified in the colon where their biologic significance remains unclear. We aimed here to characterize the motilin receptors of the rabbit colon. Distal colon and duodenum were obtained from sacrificed rabbits. Tissues homogenized by Polytron were submitted to differential centrifugation to obtain neural synaptosomes or smooth muscle plasma membranes enriched solutions. Motilin binding to these membranes was determined by the displacement of (125)I MOT by the native peptide MOT 1-22, or by peptide analogues MOT 1-12 [CH(2)NH](10-11) or GM-109 and by erythromycin derivative GM-611. Motilin binding capacity was maximum in colon nerves (49.5 +/- 6.5 fmol/mg protein vs. 19.9 +/- 2.5 in colon muscles or 9.4 +/- 2.8 and 6.6 +/- 1.2 in duodenal muscles and antral nerves respectively); all tissues expressed similar affinity for MOT 1-22, and the motilin agonist GM-611 bound equally to neural or muscle tissues from the rabbit colon; the synthetic antagonist MOT 1-12 [CH(2)NH](10-11) showed greater affinity for colon nerves than for colon muscles (plC50: 7.23 +/- 0.07 vs. 6.75 +/- 0.03). Similar results were obtained with the peptide antagonist GM-109; receptor affinity toward MOT 1-12 [CH(2)NH(10-11)] was always five times superior in neural tissues, whether they came from the colon or the antrum, than in muscle tissues, whether they were obtained from colon or from duodenum. Motilin receptors are found in very high concentration in nerves and in muscles from rabbit colon; specific motilin receptor subtypes are identified in nerves (N) and muscles (M) of the rabbit colon; N and M receptor subtypes seem independent of the organ location.

[Rehabilitation and the socio-occupational reintegration of workers who have had a myocardial infarct: a pilot study]. / La réadaptation et la réinsertion socio-professionnelle des travailleurs ayant subi un infarctus du myocarde: étude pilote.

OBJECTIVE: To evaluate in a pilot study some psychosocial impacts of a multidisciplinary rehabilitation program on work reintegration of postmyocardial infarction (MI) patients. PATIENTS AND METHODS: Two groups of 19 workers among public and governmental enterprises of the greater Quebec city area admitted to hospital for uncomplicated MI where recruited for the study. One group received multidisciplinary cardiac rehabilitation intervention and a structured return to work program. The second group received the usual care post-MI (no organized rehabilitation program). Over a period of 15 months, the following variables were measured patients' perception of their loss of autonomy, and loss of capacity to manage their emotions, physical work capacity and their capacity to meet work requirements. The impact of the intervention on convalescence time and patients' stability at work were also evaluated. RESULTS: The rehabilitation group showed a better development than the usual care group of capacity to manage emotions (P=0.004) and of capacity to return to work (P=0.006). Furthermore, the patients in the intervention group reintegrated their work environment following a convalescence of 84.32+/-35.39 days compared with 151.39 +/-75.53 days for the usual care patients (P=0.0007). CONCLUSION: A multidisciplinary rehabilitation program and a structured return to work program are beneficial for sociovocational reintegration of post-MI patients.

Monoclonal antibody production in murine ascites. I. Clinical and pathologic features.

BACKGROUND AND PURPOSE: Murine ascites production has been associated with appreciable morbidity and mortality, thus raising animal-welfare concerns. To address these concerns, the clinicopathologic changes associated with in vivo production of monoclonal antibodies in mice were characterized, and results were compared among cell lines. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally; 12 mice were sham treated (controls). Ascites fluid was collected a maximum of three times by abdominal paracentesis. Clinical observations and pre- and postabdominal tap body weights were recorded. Necropsies were performed on all mice. RESULTS: For all groups combined, overall survival to tap 1 was 98%, to tap 2 was 96%, and to tap 3 was 79%; survival among groups ranged from 90 to 100% for tap 1, 85 to 100% for tap 2, and 35 to 100% for tap 3. Disseminated intra-abdominal seeding with irregular soft tissue and/or solid tumor masses was observed at necropsy. CONCLUSIONS: Significant clinicopathologic changes were associated with monoclonal antibody production in mice, and differences between various hybridoma cell lines were apparent.

Monoclonal antibody production in murine ascites. II. Production characteristics.

OBJECTIVE: To characterize monoclonal antibody production parameters of five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample. RESULTS: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Antibody production was greatest at tap 1 for Groups 2B11 and 2C6D9; tap 2 for Group 3C9; and tap 3 for Groups RMK and 3D6. Total antibody production ranged from 422.90 to 996.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1, 2, and 3 ranged from 2.50 to 15.03 mg/ml among cell lines. CONCLUSION: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites.

Regulation of motilin release: studies with ex vivo perfused canine jejunum.

The regulatory process of motilin release was studied in segments of canine jejunum isolated and perfused ex vivo. The secretion of motilin in the effluent venous system of the isolated intestine was measured by radioimmunoassay in response to various pharmacological agents injected intra-arterially. Muscarinie agonist and antagonist, respectively, increased and decreased the release of motilin. The stimulatory effect of carbachol was still documented after tetrodotoxin (10(-5) M) was injected in the system to block neural influence on M cells. Bombesin and morphine also increased the release of motilin. The effect of bombesin was still documented in the presence of atropine or tetrodotoxin, but the stimulatory morphine effect was blocked by atropine. Both phenylephrine and octreotide decreased the release of motilin stimulated by carbachol in a jejunal segment pretreated and denervated with tetrodotoxin. Therefore, a revised model for the regulation of motilin release from M cells of intestinal mucosa can now be proposed. Cholinergic and bombesin receptors are present on M cells to encode a stimulatory signal, whereas adrenergic and somatostatin receptors are responsible for inhibitory transmission. The stimulatory effect of morphine is mediated via a muscarinic transmitter.

Multiparametric analysis of waterline contamination in dental units.

Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits. Systematic sampling of 121 dental units located at the dental school of Université de Montréal showed that none of the waterlines was spared from bacterial contamination. Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge. Differences were also found between water from the turbine and the air/water syringe. Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%). In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines. Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates. P. aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested. Dental units contaminated by P. aeruginosa showed significantly higher total bacterial counts than the others. By comparison, P. aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines. In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels. The clinical significance of these findings in relation to routine dental procedures is discussed.

Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production.

The objective of this study was to compare monoclonal antibody production in hollow fiber bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. Three hybridoma cell lines were grown in each of three different hollow fiber bioreactor systems and in groups of 20 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10(6) hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Ascites volumes and daily clinical observations were recorded. Bioreactors were harvested three times weekly for 65 day and were monitored by cell counts, cell viability and media glucose consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 455 mg vs. 168 mg; cell line 3C9, 446 mg vs. 565 mg; and cell line RMK, 997 mg vs. 1023 mg. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, and from 0.71 to 11.10 mg/ml in hollow fiber bioreactor system. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber bioreactor system merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

Gastric toxoplasmosis in acquired immunodeficiency syndrome: antemortem diagnosis with histopathologic characterization.

Gastrointestinal symptoms attributable to Toxoplasma gondii infection are distinctly unusual, and antemortem diagnosis of gastrointestinal involvement is rarely documented, particularly in the absence of cerebral manifestations or disseminated disease. This case report describes a rare example of T. gondii infection of the stomach diagnosed antemortem in a 22-year-old Haitian woman with acquired immunodeficiency syndrome (AIDS) who presented with fever and abdominal pain. An abdominal computerized tomographic scan showed thickened gastric walls. Endoscopy showed diffusely thickened gastric folds and a fundic ulcer along the greater curvature. Light and electron-microscopic examination of gastric mucosal biopsy specimens showed active Toxoplasma infection with necrosis and intracellular trophozoites within the gastric epithelium, smooth muscle cells, macrophages, and endothelial cells. Both true cysts and pseudocysts were seen. Disseminated disease was documented by the growth of T. gondii in a tissue culture from a venous blood sample. It is concluded that some patients with AIDS, particularly those from areas endemic for Toxoplasma infection, can manifest disseminated disease in unusual locations such as the gastrointestinal tract. Documentation of active T. gondii infection based on tissue cultures of venous blood or on biopsy specimens of symptomatic extracerebral sites can lead to a rapid diagnosis of toxoplasmosis, a treatable disease.

Heterogeneity of motilin receptors in the gastrointestinal tract of the rabbit.

Motilin, a 22-amino acid peptide synthesized in endocrine cells of intestinal mucosa, stimulates GI smooth muscle contractility. To elucidate the mode of action of motilin, we attempted to determine whether motilin receptors are localized on nerve cells or on smooth muscle cells of the GI tract. Mucosa-free tissues from rabbit antrum and duodenum were homogenized separately with a Polytron prior to differential centrifugation to obtain synaptosome or plasma membrane-enriched fractions, as determined by the distribution of [3H]saxitoxin (SAX) binding (neural membranes) and 5' nucleotidase (5'N) activity (smooth muscle plasma membranes). Motilin binding was evaluated by the displacement of [125I]motilin by motilin (1-22) on the various membrane fractions. In the antrum, motilin binding was highly correlated with SAX binding (r = 0.81, p < 0.0005), and also significantly with 5'N activity (r = 0.54, p < 0.05). In the duodenum, motilin binding correlated significantly with 5'N activity (r = 0.67, p < 0.005), but not with SAX binding (r = -0.11, NS). Receptor affinity, for the motilin antagonist MOT(1-12)[CH2NH]10-11, for motilin(1-22), and for the motilin agonist erythromycin lactobionate was significantly (p < 0.001, p < 0.001, and p < 0.05, respectively) higher in SAX-enriched fractions from the antrum than in 5'N-enriched fractions from the duodenum. Therefore, in the rabbit: 1) motilin receptors appear to be predominantly located on nerve tissues in the antrum and restricted to smooth muscle cells in the duodenum, and 2) antral receptors and duodenal receptors displayed different pharmacological characteristics, probably corresponding to two specific and heterogeneous motilin receptor subtypes.

Determination of 8-oxoguanine in DNA by gas chromatography--mass spectrometry and HPLC--electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography--mass spectrometry assay.

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.