Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest.
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Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the alpha/beta hydrolase-fold family and a "cap" domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model. (+info)
Digestion and absorption of bovine milk xanthine oxidase and its role as an aldehyde oxidase.
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The effects of acidic and intestinal proteolytic environments on bovine milk xanthine oxidase (XO) activity were determined in order to evaluate the extent to which this enzyme was absorbed in biologically active form. The inhibition of XO by folic acid and the relative affinities of XO for the oxidation of palmitaldehyde, stearaldehyde, and xanthine were compared. The effects of acid and gastric juice on XO activity were measured by incubating purified enzyme, and non-purified enzyme (milk), in buffers ranging in pH from 2 to 9. Fresh gastric juice was also incubated with milk. Increasing amounts of the enzyme were inactivated as the pH of the incubation mixture was reduced below pH 6.5. Below pH 3.5, the enzyme was completely inactivated. Gastric juice, pH juice incubated with milk. Milk XO activity was reduced 36% when mild was incubated with an equal volume of gastric juice. Homogenized milk had 59% less XO activity compared with raw molk. Fresh raw milk XO, homogenized milk XO, and purified XO were equally susceptible to inactivation by acid or gastric juice. After incubation of milk with gastric juice, or gastric juice followed by pancreatin, XO activity was associated with a macromolecule of 300,000 daltons molecular weight and subunits containg activity were not found. It was estimated that 0.00008% of the XO in the intestine was absorbed. Both folic acid and allopurinol inhibited XO activity in vitro. Allopurinol was 3.5 times more potent an inhibitor than folic acid. A large excess of dietary folic acid did not reduce rat liver or intestinal XO activity in vivo. XO had a much greater affinity for xanthine than for palmitaldehyde or stearaldehyde substrates. It was estimated that of 100 mg of XO in fresh raw milk, 41 mg remained after homogenization, 27 mg entered the intestine and only 20 ng were absorbed as intact enzyme. (+info)
Ability of mosapride to bind to 5-HT4 receptor in the human stomach.
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Ability of mosapride, a gastrokinetic agent, to bind to 5-HT4 receptor was examined in the stomach of human and guinea pig by in vitro receptor autoradiography. [125I]SB207710 binding sites were detected in the muscle layer including the myenteric plexus of the stomach from both humans and guinea pigs, although the binding was observed more clearly and densely in the stomach of guinea pigs than humans. Mosapride as well as SB204070 inhibited the binding of [125I]SB207710. Thus, mosapride possesses the ability to bind to 5-HT4 receptors of human stomach and may modulate the motility, as in the case of guinea pig stomach. (+info)
Gut vagal afferent lesions increase meal size but do not block gastric preload-induced feeding suppression.
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Subdiaphragmatic vagal afferent (SVA) signals arising from gut sites may provide critical feedback for the control of food intake within a meal. To evaluate the role of SVAs in both spontaneous and scheduled meals, food intake was assessed in two paradigms in male Sprague-Dawley rats. In the first study, control (Con) rats (n = 6) and rats with subdiaphragmatic vagal deafferentation (SDA) (n = 7) had 12-h nightly access to Ensure liquid diet (1 kcal/ml). SDA rats had larger and fewer meals and maintained initial rapid rates of licking, yet total numbers of licks were unaffected. In the second study, Con (n = 8) and SDA (n = 7) rats had scheduled access to 12. 5% liquid glucose after overnight food deprivation. Glucose intake was assessed after 5-ml gastric preloads of 0.9% saline or glucose, peptone, and Intralipid solutions at three concentrations (0.5, 1, and 2 kcal/ml). Glucose and peptone preloads suppressed intake similarly in Con and SDA rats, whereas Intralipid was ineffective. These results suggest that meal-related SVA signals 1) are not critical in determining preload-induced feeding suppression after deprivation, yet 2) contribute to satiety during spontaneous meals. (+info)
HCO-3 reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response.
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Mice with a targeted disruption of Na+/H+ exchanger NHE-3 gene show significant reduction in HCO-3 reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum HCO-3, however, is only mildly decreased (P. Schulties, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998), indicating possible adaptive upregulation of HCO-3-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 -/-) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2 (net HCO-3 flux, JtCO2) was measured in the presence of 10 microM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 microM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2 increased in NHE-3 mutant mice (3.42 +/- 0.28 in WT to 5.71 +/- 0.39 pmol. min-1. mm tubule-1 in mutants, P < 0.001). The SCH-sensitive net HCO-3 flux remained unchanged, whereas the DES-sensitive HCO-3 flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2 increased in NHE-3 mutant mice (8.8 +/- 0.7 in WT to 14.2 +/- 0.6 pmol. min-1. mm tubule-1 in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive HCO-3 fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl-/HCO-3 exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that HCO-3 reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory HCO-3 reabsorption in NHE-3-deficient animals. (+info)
Feeding difficulties and foregut dysmotility in Noonan's syndrome.
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PURPOSE: Noonan's syndrome is a common dysmorphic syndrome in which failure to thrive and gastrointestinal symptoms are frequent but poorly understood. DESIGN: Twenty five children with Noonan's syndrome were investigated by contrast radiology, pH monitoring, surface electrogastrography (EGG), and antroduodenal manometry (ADM). RESULTS: Sixteen had poor feeding and symptoms of gastrointestinal dysfunction. All 16 required tube feeding. Seven of 25 had symptoms of foregut dysmotility and gastro-oesophageal reflux. In the most symptomatic children (four of seven) EGG showed fasting frequency gradient loss along the stomach fundus and pylorus with antral postprandial frequency loss. ADM showed shortened fasting cycle length, with abnormal phase III and shortened postprandial activity containing phasic contractions. IMPLICATIONS: Gastroduodenal motor activity was reminiscent of 32-35 week preterm patterns. The feeding difficulties appear to resolve as gut motility matures. In Noonan's syndrome, feeding problems appear to be the result of delayed gastrointestinal motor development. (+info)
Interstitial cells of cajal generate electrical slow waves in the murine stomach.
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1. The gastric corpus and antrum contain interstitial cells of Cajal (ICC) within the tunica muscularis. We tested the hypothesis that ICC are involved in the generation and regeneration of electrical slow waves. 2. Normal, postnatal development of slow wave activity was characterized in tissues freshly removed from animals between birth and day 50 (D50). Slow wave amplitude and frequency increased during this period. Networks of myenteric ICC (IC-MY) were present in gastric muscles at birth and did not change significantly in appearance during the period of study as imaged by confocal immunofluorescence microscopy. 3. IC-MY networks were maintained and electrical rhythmicity developed in organ culture in a manner similar to normal postnatal development. Electrical activity was maintained for at least 48 days in culture. 4. Addition of a neutralizing antibody (ACK2) for the receptor tyrosine kinase, Kit, to the culture media caused progressive loss of Kit-immunoreactive cells. Loss of Kit-immunoreactive cells was associated with loss of slow wave activity. Most muscles became electrically quiescent after 3-4 weeks of exposure to ACK2. 5. In some muscles small clusters of Kit-immunoreactive IC-MY remained after culturing with ACK2. These muscles displayed slow wave activity but only in the immediate regions in which Kit-positive IC-MY remained. These data suggest that regions without Kit-immunoreactive cells cannot generate or regenerate slow waves. 6. After loss of Kit-immunoreactive cells, the muscles could not be paced by direct electrical stimulation. Stimulation with acetylcholine also failed to elicit slow waves. The data suggest that the generation of slow waves is an exclusive property of IC-MY; smooth muscle cells may not express the ionic apparatus necessary for generation of these events. 7. We conclude that IC-MY are an essential element in the spontaneous rhythmic electrical and contractile activity of gastric muscles. This class of ICC appears to generate slow wave activity and may provide a means for regeneration of slow waves. (+info)
Histological and electron-microscopic observations on the mucosa of pediculated duodenal wall graft transplanted to the stomach in Wistar rats.
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OBJECTIVE: To study the mechanism of gastric metaplasia in duodenum through the transplantation of a flap of duodenal wall with vascular pedicle to the stomach. METHODS: The pediculated duodenal wall flaps of Wistar rats were transplanted to their stomachs. The rats were killed at the 3rd, 6th, 9th and 12th month after the operation respectively, and histological changes of the duodenal grafts were observed with optical and electron microscopy. RESULTS: Gastric metaplasia was found in the mucosa of duodenal grafts transplanted to the stomach at the 6th, 9th and 12th month. CONCLUSIONS: The formation of gastric metaplasia in the duodenal mucosa may be related to a change of the microenvironment around the tissues, and duodenal mucosa may differentiate into gastric epithelium by the decrease of pH value. (+info)