Topology, subcellular localization, and sequence diversity of the Mlo family in plants.
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Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors. (+info)
Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.
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In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo. (+info)
Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels.
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Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3-3.5 wk of age, 10-12 g) gained 49 +/- 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 +/- 4%, AQP1 null mice gained only 4 +/- 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [(14)C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice. (+info)
Identification of a nitric oxide-regulated zinc finger containing transcription factor using motif-directed differential display.
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We report here the isolation of human zinc finger 2 (HZF2), a putative zinc-finger transcription factor, by motif-directed differential display of mRNA extracted from histamine-stimulated human vein endothelial cells. The expression of HZF2 mRNA in venous endothelial cells was verified by Northern blot analysis, which also revealed an enrichment of HZF2 mRNA in lymphocytes and monocytes. Histamine induced a time- and concentration-dependent upregulation of HZF2 level with a 6-fold peak increase of mRNA at 30 min. HZF2 upregulation was abolished by different NOS isozyme inhibitors. Guanylate cyclase inhibition resulted in a significant decrease of HZF2 expression. These observations indicate HZF2 as a potentially interesting new target for studies concerning rapid NO-mediated gene regulation. (+info)
Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist-driven internalization, in contrast to the NPY Y2 receptor.
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Guinea-pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 degrees C, and essentially absent at or below 15 degrees C, possibly in connection to the large molecular size of the receptor-ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 degrees C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes. (+info)
Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability.
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It is widely accepted that solvent-exposed sites in proteins play only a negligible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T(den) difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2 degrees C at pH 2.0. The DeltaH(den) versus T(den) plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal* mole(-1) * K(-1); 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal*mole(-1). Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) =7.1 degrees C). Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution. (+info)
Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat.
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Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity. (+info)
Efficacy of oral pancreatic enzyme therapy for the treatment of fat malabsorption in HIV-infected patients.
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BACKGROUND: Nutrient malabsorption is a negative prognostic factor in acquired immunodeficiency syndrome and recent studies have shown that pancreatic insufficiency is a codetermining factor of malabsorption. AIMS: To evaluate the effectiveness of open-label oral pancreatic enzyme supplementation therapy in acquired immunodeficiency syndrome patients with fat malabsorption. PATIENTS AND METHODS: Twenty-four consecutive patients with human immunodeficiency virus infection and fat malabsorption were recruited (11 males, 13 females; median age, 9.1 years). Faecal fat loss was evaluated by steatocrit assay at entry to the study (T-0), after 2 weeks (T-1) without pancreatic enzyme treatment and after a further 2 weeks (T-2) of treatment with pancreatic extracts (Creon 10 000 at a dose of 1000 units of lipase per gram of ingested dietary fat). Faecal elastase-1 and chymotrypsin were assayed at entry. RESULTS: Six patients (25%) had abnormally low elastase-1 and/or chymotrypsin faecal concentration. In all patients, steatocrit values were elevated at both T-0 and T-1. Five patients proved intolerant to pancreatic enzyme treatment because of the onset of abdominal pain, and therapy was discontinued. In the 19 patients who concluded the study, steatocrit values during pancreatic enzyme treatment (T-2) were significantly lower than at entry (P < 0.0001). At T-2, in eight of 19 patients, steatocrit values were within the normal limit and the frequency of cases cured or improved on pancreatic enzyme therapy (at T-2) was significantly higher than that observed during the previous study period without enzyme treatment (T-1) (P < 0.01). A positive significant correlation was found between steatocrit values at entry and the Centers for Disease Control class (P < 0.0005); also, the decrease in steatocrit values during pancreatic enzyme therapy (difference between steatocrit value at T-2 and steatocrit value at T-0) positively correlated with the Centers for Disease Control class (P < 0.05). CONCLUSIONS: This pilot, open-label study showed that pancreatic enzyme supplementation therapy is highly effective in reducing faecal fat loss in human immunodeficiency virus-infected patients with nutrient malabsorption. Further double-blind studies must be undertaken to verify these results and, if they are confirmed, pancreatic enzymes can be added to our weapons in the fight against human immunodeficiency virus-associated nutrient malabsorption. (+info)