In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gramnegative bacteria, whereas PGRP-LE binds to the DAPtype peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.
We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.
The permeation of ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), a novel ACTH analogue, across the intestinal mucosae has been examined by use of isolated intestinal membranes from rats in a modified Ussing chamber. Regional differences were observed in the permeation of ebiratide across intestinal membranes; the order of membrane permeability was jejunum > ileum > duodenum > colon. Overall, the permeation of ebiratide was relatively poor. The effects of various absorption enhancers were examined to increase the intestinal permeability to ebiratide. Sodium glycocholate and sodium caprate had no significant enhancing effect on the permeability of the jejunal membrane, but significantly enhanced the permeation of ebiratide through the colonic membrane. On the other hand, N-dodecyl-beta-D-maltopyramoside (LM) significantly enhanced the permeation of ebiratide through both jejunal and colonic membranes. In general, the absorption-enhancing effects of these agents were more predominant in the colon than in the jejunum. Membrane damage by the absorption enhancers was evaluated by measuring the amount of protein released from the intestinal membrane. It was found that all the absorption enhancers slightly increased the amount of protein released, but that the amounts of protein released in the presence of these enhancers were much less than in the presence of ethylenediaminetetraacetic acid (EDTA), used as a positive control. These findings suggest that the absorption enhancers, especially LM might be useful adjuvants for improving the intestinal absorption of peptide and protein drugs, including ebiratide.
Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Galpha(q/11) and a part of P2Y(2) receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-beta-cyclodextrin (CD) disrupted not only lipid raft markers but also Galpha(q/11) and P2Y(2) receptors in this fraction. In the presence of CD, P2Y(2) receptor-mediated phosphoinositide hydrolysis and [Ca(2+)](i) elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G(q/11)-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115-regulator of G protein signaling, which inhibits the G(12/13) signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G(q/11) signaling pathway. These results suggest that lipid rafts are critical for P2Y(2) receptor-mediated G(q/11)-PLC-Ca(2+) signaling and this cascade is important for cell migration in NG 108-15 cells.
The effects of various protease inhibitors on the stability of leucine enkephalin (Leu-Enk) and [D-Ala2,D-Leu5] enkephalin (DADLE) were investigated, and the permeability of these peptides was also examined in an in vitro Ussing chamber. Captopril, thiorphan, bacitracin, bestatin, puromycin, amastatin, and sodium glycocholate (Na-GC) were chosen as protease inhibitors. Regional differences in the stability of Leu-Enk and DADLE were observed, and the rank order of the stability of these peptides was colon > duodenum > ileum > jejunum. Na-GC, amastatin, and puromycin were effective protease inhibitors for improving the stability of these peptides, although captopril and thiorphan did not improve the stability of Leu-Enk. In the transport studies, Leu-Enk did not cross the intestinal membrane in the absence of protease inhibitors, but its transport was improved in the presence of Na-GC. In addition, Na-GC, amastatin, and puromycin improved the permeability of DADLE in both jejunum and colon, while the permeability of DADLE was not improved by the addition of captopril, thiorphan, and bestatin. Furthermore, the permeability of 6-carboxyfluorescein, a poorly absorbable and stable compound, was also improved in the presence of Na-GC and bacitracin at a concentration of 10 mM. These findings indicated that amastatin, puromycin, and Na-GC at a concentration of 0.5 mM might increase the permeability of DADLE due to the improved stability of DADLE in the donor site. However, Na-GC and bacitracin at a concentration of 10 mM had absorption-enhancing activities which might be also related to the enhanced permeability of DADLE across the intestinal membrane.
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