Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA. Effect of Aedes trypsin modulating oostatic factor on trypsin biosynthesis.
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Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3' and 5' ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-dimensional model of Diaprepes trypsin contained two domains of beta-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28-100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor-like signal. (+info)
Outbreak of Salmonella serotype Muenchen infections associated with unpasteurized orange juice--United States and Canada, June 1999.
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During June 1999, Public Health-Seattle and King County (PHSKC) and the Washington state health department and the Oregon Health Division independently investigated clusters of diarrheal illness attributed to Salmonella serotype Muenchen infections in each state. Both clusters were associated with a commercially distributed unpasteurized orange juice traced to a single processor, which distributes widely in the United States. As of July 13, 207 confirmed cases associated with this outbreak have been reported by 15 states and two Canadian provinces; an additional 91 cases of S. Muenchen infection reported since June 1 are under investigation. This report summarizes the two state-based investigations and presents preliminary information about the outbreak in the other states and Canada. (+info)
A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa.
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A simplified protocol of subtractive hybridization based on the technique of L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt (1985, Proc Natl Acad Sci USA, 82, 4778-4782) was used to obtain DNA sequences specific to Xylella fastidiosa isolated from diseased citrus plants. As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: Xy. fastidiosa 788 isolated from another host (plum), Xanthomonas campestris pv. campestris and Burkholderia gladioli strains. A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to Xy. fastidiosa from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia coli serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy. fastidiosa DNA. These primers detected Xy. fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Erwinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, P. cichorii, Pseudomonas syringae pv. garcae). The isolation of a DNA fragment specific to Xy. fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other micro-organisms. (+info)
Citric acid or orange juice for the 13C-urea breath test: the impact of pH and gastric emptying.
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BACKGROUND: There is an ongoing debate about the optimal test drink to be used in the 13C-urea breath test (13C-UBT). We recently reported that a citric acid solution is the optimal test drink in the 13C-UBT, because it provides a high 13CO2 recovery and the excellent accuracy of the test appears optimal compared to other test meals. Orange juice, because of a better taste, is also propagated as a test drink in the 13C-UBT. AIM: To compare the diagnostic accuracy of the 13C-UBT with either orange juice or citric acid solution as a test drink. Furthermore, the effect of these test drinks on the gastric emptying rate was determined. METHODS: H. pylori status was assessed by histology, rapid urease test and culture in 50 consecutive dyspeptic patients. A 13C-UBT was performed on two consecutive days by giving 75 mg of 13C-urea randomly dissolved in 200 mL 0.1 M citric acid solution or 200 mL orange juice. The 13CO2/12CO2 ratio was measured in breath samples taken before and 15, 30, 45 and 60 min after administration of the test drink. The gastric emptying rate of orange juice and citric acid solution was compared to that of water in 10 healthy subjects on three consecutive days by means of a 13C-sodium acetate breath test; 50 mg of 13C-sodium acetate was dissolved in 200 mL of each solution and breath samples were collected before and every 10 min for 90 min after administration of the test drink. RESULTS: Twenty-six out of 50 patients (52%) were infected with H. pylori. Significantly higher values over baseline (35.7+/-5.2 per thousand vs. 23.2+/-3.4 per thousand, P<0.001) and higher area under the curve (1507+/-198 vs. 927+/-128, P<0.001) were observed in H. pylori-positive patients when citric acid solution was administered compared with orange juice. Sensitivity of the 13C-UBT was 100% when citric acid was used as a test drink and 88% with orange juice. Specificity was 100% with both test drinks. Gastric emptying of citric acid solution (t1/2 = 60.9+/-3.5 min) was significantly slower than that of orange juice (t1/2 = 49.7+/-3.1 min, P<0.001). CONCLUSION: 13C-UBT loses diagnostic accuracy when orange juice instead of citric acid is used as a test drink. The faster gastric emptying of orange juice might be responsible for the lower diagnostic accuracy of the 13C-UBT. (+info)
Role of olfaction in food preference as evaluated in an animal model.
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Food preference in individual animals is regulated by brain activity. Two murine model systems for investigating food preference were developed by focusing on fruit juices. In a home-cage, two-bottle test, the volume of apple juice consumed was found to be much larger than that of orange juice. In a two-nozzle "Drinkometer" test, by which each mouse was kept in a 38 cm (W) x 32 cm (D) cage and each drinking event was recorded by an electronic "Drinkometer" device, it was again found that the mice preferred drinking apple juice to orange juice. To elucidate the role of olfaction in this food preference, mice were subjected to an olfactory bulbectomy to remove the olfaction capability. In the home-cage two-bottle test, the preference for apple juice over orange juice was apparent even after the olfactory bulbectomy, indicating that olfaction was not essential for the formation of food preference behavior. In contrast, in the two-nozzle "Drinkometer" test, the preference for apple juice over orange juice was found to be abrogated by this surgery, implying the involvement of olfaction-based memory on food preference behavior. (+info)
Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by grapefruit juice components.
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AIMS: Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are both expressed in the intestinal mucosa and present a barrier to oral drug delivery. CYP3A4 and P-gp share both overlapping tissue distribution and substrate specificity. Grapefruit juice interactions with CYP3A4 substrates are well documented and occur as a consequence of down regulation of intestinal CYP3A4. The aim of the present study was to screen grapefruit juice components against the CYP3A4-mediated metabolism and P-gp mediated transport of the HIV-1 protease inhibitor saquinavir. METHODS: Five grapefruit juice components: quercetin, naringin, naringenin, 6', 7'-dihydroxybergamottin and bergamottin were screened as potential inhibitors of the metabolism of saquinavir by human liver microsomes. The known CYP3A4 inhibitor ketoconazole was also screened for inhibitory potential. These compounds were also screened as modulators of P-gp activity by assessing the directional transport of saquinavir across Caco-2 cell monolayers which express P-gp. The effect of verapamil, a known modulator of P-gp function, was also determined in these cell lines. RESULTS: On preincubation, 6', 7'-dihydroxybergamottin and bergamottin inhibited the metabolism of saquinavir, with IC50 values of 0.33+/-0.23 muM and 0.74+/-0.13 muM, respectively (n=3). Ketoconazole achieved an IC50 of 0. 55+/-0.12 muM (n=4). The other compounds studied failed to reach IC50 at concentrations of up to 100 muM. The transport of saquinavir in the basolateral-->apical (BL-->AP) direction exceeded that in the apical -->basolateral direction (AP-->BL), with apparent permeability coefficients of 199.2+/-15.8x10-7 cm s-1 and 8.00+/-1. 13x10-7 cm s-1, respectively (n=3) which is indicative of a polarized efflux mechanism. The ratio of BL-->AP/AP-->BL for saquinavir was 25, but in the presence of verapamil and ketoconazole this ratio was reduced to 3.6 and 4.0, respectively (n=3), indicating extensive inhibition of P-gp mediated saquinavir efflux. Of the grapefruit juice components studied only naringin and 6', 7'-dihydroxybergamottin had any appreciable effect, reducing the ratio to 7.6 and 7.1, respectively (n=3); but this was due solely to increased AP-->BL transport. CONCLUSIONS: Grapefruit juice components inhibit CYP3A4-mediated saquinavir metabolism and also modulate, to a limited extent, P-gp mediated saquinavir transport in Caco-2 cell monolayers. The in vivo effects of grapefruit juice coadministration are most likely the result of effects on CYP3A4 (inhibition and down regulation) and only to a minor extent on modulation of P-gp function. (+info)
Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker.
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A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri. (+info)
Effect of diclofenac, disulfiram, itraconazole, grapefruit juice and erythromycin on the pharmacokinetics of quinidine.
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AIMS: In vitro studies suggest that the oxidation of quinidine to 3-hydroxyquinidine is a specific marker reaction for CYP3A4 activity. To assess the possible use of this reaction as an in vivo marker of CYP3A4 activity, we studied the involvement of cytochromes CYP2C9, CYP2E1 and CYP3A4 in the in vivo oxidative metabolism of quinidine. METHODS: An open study of 30 healthy young male volunteers was performed. The pharmacokinetics of a 200 mg single oral dose of quinidine was studied before and during daily administration of 100 mg diclofenac, a CYP2C9 substrate (n=6); 200 mg disulfiram, an inhibitor of CYP2E1 (n=6); 100 mg itraconazole, an inhibitor of CYP3A4 (n=6); 250 ml single strength grapefruit juice twice daily, an inhibitor of CYP3A4 (n=6); 250 mg of erythromycin 4 times daily, an inhibitor of CYP3A4 (n=6). Probes of other enzyme activities, caffeine (CYP1A2), sparteine (CYP2D6), mephenytoin (CYP2C19), tolbutamide (CYP2C9) and cortisol (CYP3A4) were also studied. RESULTS: Concomitant administration of diclofenac reduced the partial clearance of quinidine by N-oxidation by 27%, while no effect was found for other pharmacokinetic parameters of quinidine. Concomitant administration of disulfiram did not alter any of the pharmacokinetic parameters of quinidine. Concomitant administration of itraconazole reduced quinidine total clearance, partial clearance by 3-hydroxylation and partial clearance by N-oxidation by 61, 84 and 73%, respectively. The renal clearance was reduced by 60% and the elimination half-life increased by 35%. Concomitant administration of grapefruit juice reduced the total clearance of quinidine and its partial clearance by 3-hydroxylation and N-oxidation by 15, 19 and 27%, respectively. The elimination half-life of quinidine was increased by 19%. The caffeine metabolic index was reduced by 25%. Concomitant administration of erythromycin reduced the total clearance of quinidine and its partial clearance by 3-hydroxylation and N-oxidation by 34, 50 and 33%, respectively. Cmax was increased by 39%. CONCLUSIONS: The results confirm an important role for CYP3A4 in the oxidation of quinidine in vivo, and this applies particularly to the formation of 3-hydroxyquinidine. While a minor contribution of CYP2C9 to the N-oxidation of quinidine is possible, a major involvement of the CYP2C9 or CYP2E1 enzymes in the oxidation of quinidine in vivo is unlikely. (+info)