A continuous spectrophotometric assay for the determination of diamondback moth esterase activity

Conventional methods to determine esterase activity from insects are composed of a three-step process where the enzyme is allowed to hydrolyze a 1-naphthyl acetate substrate, that reaction is quenched by a SDS detergent, and then a Fast Blue B dye complex is formed with 1-naphthol, the product of 1-naphthyl acetate hydrolysis. These methods measure dye-product complex rather than the product, 1-naphthol. A new assay is presented that continuously monitors the formation of 1-naphthol with the hydrolysis of an esterase substrate. The esterase activity was determined as the slope of the linear regression change in absorbance over time at 320 nm. The continuous assay provides a simple, rapid, and sensitive method for measuring esterases extracted from a single diamondback moth in 1-10 min. The detection limit of the assay is approximately 0.6 microM 1-naphthol. The 1-naphthol product from the esterase reaction was confirmed by HPLC analysis. According to the assay, the K(m) and V(max) values of the esterase were 28 +/- 2 microM and 6.0 +/- 0.1 microM/min, respectively, at 37 degrees C for 1-naphthyl acetate. The K(i) value was 9 +/- 2 microM using azadirachtin, an insecticide from neem tree, Azadirachta indica (A.Juss). Azadirachtin was a reversible competitive inhibitor of the esterase activity.     

Erythropoietin protects the intestine against ischemia/ reperfusion injury in rats

Previous studies have shown that erythropoietin (EPO) has protective effects against ischemia/reperfusion (I/R) injury in several tissues. The aim of this study was to determine whether EPO could prevent intestinal tissue injury induced by I/R. Wistar rats were subjected to intestinal ischemia (30 min) and reperfusion (60 min). A single dose of EPO (5000 U/kg) was administered intraperitoneally at two different time points: either at five minutes before the onset of ischemia or at the onset of reperfusion. At the end of the reperfusion period, jejunum was removed for examinations. Myeloperoxidase (MPO), malondialdehyde (MDA), and antioxidant defense system were assessed by biochemical analyses. Histological evaluation was performed according to the Chiu scoring method. Endothelial nitric oxide synthase (eNOS) was demonstrated by immunohistochemistry. Apoptotic cells were determined by TUNEL staining. Compared with the sham, I/R caused intestinal tissue injury (Chiu score, 3+/-0.36 vs 0.4+/-0.24, P<0.01) and was accompanied by increases in MDA levels (0.747+/-0.076 vs 0.492+/-0.033, P<0.05), MPO activity (10.51+/-1.87 vs 4.3+/-0.45, P<0.05), intensity of eNOS immunolabelling (3+/-0.4 vs 1.3+/-0.33, P<0.05), the number of TUNEL-positive cells (20.4+/-2.6 vs 4.6+/-1.2, P<0.001), and a decrease in catalase activity (16.83+/-2.6 vs 43.15+/-4.7, P<0.01). Compared with the vehicle-treated I/R, EPO improved tissue injury; decreased the intensity of eNOS immunolabelling (1.6+/-0.24 vs 3+/-0.4, P<0.05), the number of TUNEL-positive cells (9.2+/-2.7 vs 20.4+/-2.6, P<0.01), and the high histological scores (1+/-0.51 vs 3+/-0.36, P<0.01), and increased catalase activity (42.85+/-6 vs 16.83+/-2.6, P<0.01) when given before ischemia, while it was found to have decreased the levels of MDA (0.483+/-0.025 vs 0.747+/-0.076, P<0.05) and MPO activity (3.86+/-0.76 vs 10.51+/-1.87, P<0.05), intensity of eNOS immunolabelling (1.4+/-0.24 vs 3+/-0.4, P<0.01), the number of TUNEL-positive cells (9.1+/-3 vs 20.4+/-2.6, P<0.01), and the number of high histological scores (1.16+/-0.4 vs 3+/-0.36, P<0.05) when given at the onset of reperfusion. These results demonstrate that EPO protects against intestinal I/R injury in rats by reducing oxidative stress and apoptosis. We attributed this beneficial effect to the antioxidative properties of EPO.     

Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats

The effect of an intestinal microflora consisting of selected microbial species on myoelectric activity of small intestine was studied using germ-free rat models, with recording before and after specific intestinal colonization, in the unanesthetized state. Intestinal transit, neuropeptides in blood (RIA), and neuromessengers in the intestinal wall were determined. Clostridium tabificum vp 04 promoted regular spike burst activity, shown by a reduction of the migrating myoelectric complex (MMC) period from 30.5 +/- 3.9 min in the germ-free state to 21.2 +/- 0.14 min (P < 0.01). Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11 reduced the MMC period from 27.9 +/- 4.5 to 21.5 +/- 2.1 min (P < 0.02) and accelerated small intestinal transit (P < 0.05). Micrococcus luteus showed an inhibitory effect, with an MMC period of 35.9 +/- 9.3 min compared with 27.7 +/- 6.3 min in germ-free rats (P < 0.01). Inhibition was indicated also for Escherichia coli X7 gnotobiotic rats. No consistent changes in slow wave frequency were observed. The concentration of neuropeptide Y in blood decreased after introduction of conventional intestinal microflora, suggesting reduced inhibitory control. Intestinal bacteria promote or suppress the initiation and aboral migration of the MMC depending on the species involved. Bacteria with primitive fermenting metabolism (anaerobes) emerge as important promoters of regular spike burst activity in small intestine.     

Starvation alters the activity and mRNA level of glutaminase and glutamine synthetase in the rat intestine

The metabolism of glutamine, the main respiratory fuel of enterocytes, is governed by the activity of glutaminase and glutamine synthetase. Because starvation induces intestinal atrophy, it might alter the rate of intestinal glutamine utilization. This study examined the effect of starvation on the activity, level of mRNA, and distribution of mRNA of glutaminase and glutamine synthetase in the rat intestine. Rats were randomized into groups and were either: (1) fed for 2 days with rat food ad libitum or (2) starved for 2 days. Standardized segments of jejunum and ileum were removed for the estimation of enzyme activity, level of mRNA, and in situ hybridization analysis. The jejunum of the fed rats had a greater activity of both enzymes per centimeter of intestine (P < 0.01), a greater glutaminase specific activity (1.97 +/- 0.45 vs. 1.09 +/- 0.34 micromol/hr/mg protein, P < 0.01), and a lower level of glutaminase and glutamine synthetase mRNA. The ileum of the fed rats had a greater activity of glutamine synthetase per centimeter of intestine (162.9 +/- 50.6 vs. 91.0 +/- 23.1 nmol/hr/cm bowel, P < 0.01), a lower level of glutaminase mRNA, and a greater level of glutamine synthetase mRNA. In situ hybridization analysis showed that starvation does not alter the distribution of glutaminase and glutamine synthetase mRNA in the intestinal mucosa. This study confirms that starvation decreases the total intestinal activity per centimeter of both glutaminase and glutamine synthetase. More importantly, the results indicate that the intestine adapts to starvation by accumulating glutaminase mRNA. This process prepares the intestine for a restoration of intake.     

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.     

Purification and properties of three esterases from Brevibacterium sp. R312

C. LAMBRECHTS, J. ESCUDERO AND P. GALZY. 1995. The esterases of Brevibacterium sp. R312 were found to have an intracellular location. Electrophoresis of lysed cell supernatant fluids revealed seven bands of esterase activity in the presence of α-naphthyl acetate. Eight esterases were separated by anion exchange chromatography. The three main esterases (esterase 4b, 2 and 4a) of Brevibacterium sp. R312 were purified. The molar masses, the pH optima, the temperature optima and heat stabilities were determined. Esterase 2 differed from the two others in sensitivity to inhibitors. Esterase 4b differed from esterases 2 and 4a in its substrate specificity. This enzyme hydrolyses aliphatic and nitrophenyl esters. The spectrum of activity of the two other esterases is narrower. They hydrolysed only naphthyl esters and, in the case of esterase 2, tributyrate and ethyl butyrate.     

Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization

It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 +/- 0.09 versus 1.04 +/- 0.07 micromol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 +/- 2 versus 2.4 +/- 0.3 micromol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 +/- 0.5 versus 2.6 +/- 0.0 micromol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 +/- 0.3 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 +/- 0.8 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Genetic and biochemical studies of the highly active esterases A′ and B associated with organophosphate resistance in mosquitoes of the Culex pipiens complex

The highly active esterases A' and B that cannot be dissociated from OP resistance in Culex pipiens from France and California are shown to have equivalent Km values (2.1 x 10(-6) M/min/mosquito) but different turnover rates (Vm = 2.13 and 0.57 x 10(-6) M/min/mosquito, respectively) and pH for maximum activity. Both enzymes have broad substrate specificities and at least one, esterase A', can hydrolyze OP insecticides. In addition, esterases A' and B are coded by two closely linked genes, Est-3 and Est-2, respectively (0.67 unit of crossing over), located on the same autosome as pl, a locus attributed to linkage group III. The estimated distance between Est-2 and pl was 9.4 units.     

Relationship between nociceptin/orphanin FQ and cerebral hemodynamics after hypoxia-ischemia in piglets

This study was designed to characterize the role of the newly described endogenous opioid nociceptin/orphanin FQ (NOC/oFQ) in reduced cerebral blood flow (CBF) observed after ischemia-reperfusion (I/R) and combined hypoxia and ischemia-reperfusion (H-I/R), as a function of time after onset of reperfusion in newborn pigs equipped with a closed cranial window. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, whereas hypoxia (10 min) decreased PO(2) to 35 +/- 3 mmHg with unchanged PCO(2). I/R elevated cerebrospinal fluid (CSF) NOC/oFQ from 67 +/- 4 to 266 +/- 29 pg/ml within 1 h, whereas values returned to control level within 4 h of reperfusion. H-I/R elevated CSF NOC/oFQ to 483 +/- 67 pg/ml within 1 h, and such values returned slowly to control level within 12 h of reperfusion. Topical NOC/oFQ (10(-8) M, 10(-6) M)-induced vasodilation was attenuated by I/R and reversed to vasoconstriction by H-I/R at 1 h of reperfusion (control, 9 +/- 1 and 16 +/- 1%; I/R, 3 +/- 1 and 6 +/- 1%; H-I/R, -6 +/- 1 and -11 +/- 1%). Such altered dilation returned to control values within 4 h in I/R animals and within 12 h in H-I/R animals. Blood flow in the cerebrum was reduced from 58 +/- 4 to 33 +/- 2 ml x min(-1) x 100 g(-1) within 1 h and returned to control value within 4 h in I/R animals. In animals pretreated with [F/G]NOC/oFQ(1-13)-NH(2) (1 mg/kg iv), an NOC/oFQ antagonist, however, CBF only fell to 43 +/- 3 ml x min(-1) x 100 g(-1) at 1 h of reperfusion. Similar observations were made in H-I/R animals. These data suggest that an elevated CSF NOC/oFQ concentration and altered vascular responsiveness to this opioid contribute to reductions in CBF observed after either I/R or H-I/R.     

An additional locus controlling liver esterases of chickens

Starch gel electrophoresis of liver extracts of chickens revealed six regions, designated I to VI, of esterase activity. Three phenotypes, A, B and AB, were found in esterases in region I. These phenotypes were shown to be controlled by a pair of codominant autosomal alleles, Es -5^A and Es -5^B.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Isozyme variability in species of the genus Drosophila. III. Qualitative comparison of the esterases of D. aldrichi and D. mulleri

Two closely related species, Drosophila aldrichi and D. mulleri, are compared on the basis of their esterase isozyme patterns after starch gel electrophoresis. Comparable esterases between the two species are identified by substrate specificity, inhibition, and enhancement of esterase activity by various agents. The extensive electrophoretic variability of most of the esterases in nature is described and data relating to the inheritance of the various enzyme forms are presented.This work was supported (in part) by a U.S. Public Health Service research grant (GM 11609 to W. S. Stone and M. R. Wheeler) and a training grant (2 T1-GM 337-07 to R. P. Wagner et al.) from the National Institutes of Health.     

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.     

Inhibition of human pancreatic and biliary output but not intestinal motility by physiological intraileal lipid loads

Lipid perfusion into the distal ileal lumen at supraphysiological loads inhibits pancreatic exocrine secretion and gastrointestinal motility in humans. In the present study, we sought to determine the effects of physiological postprandial intraileal lipid concentrations on endogenously stimulated pancreaticobiliary secretion, intestinal motility, and release of regulatory mediators. Eight healthy volunteers were intubated with an oroileal multilumen tube for continuous duodenal perfusion of essential amino acids (450 mumol/min), ileal perfusion of graded doses of lipids (0, 50 and 100 mg/min, each dose for 90-120 min), aspiration of duodenal and ileal chyme, and intestinal manometry. Venous blood samples were obtained for measurement of GLP-1 and PYY. Ileal lipid perfusion dose dependently decreased endogenously stimulated trypsin [262 +/- 59 vs. 154 +/- 42 vs. 92 +/- 20 U/min (P < 0.05)] and bile acid output [18.6 +/- 1.9 vs. 8.4 +/- 2.8 vs. 3.0 +/- 1.0 micromol/min (P < 0.05)]. Duodenal motor activity was not inhibited by either lipid dose. Trypsin and bile acid output correlated inversely with the release of GLP-1 and PYY (absolute value of R > 0.84; P < 0.05), whereas the motility index did not. Physiological postprandial ileal lipid concentrations dose dependently inhibited human digestive pancreatic protease and bile acid output, but not intestinal motor activity. Thus physiological postprandial ileal nutrient exposure may be of importance for the termination of digestive secretory responses. Ileocolonic release of GLP-1 and PYY appears to participate in mediating these effects.