High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.

RATIONALE: Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis. METHODS: Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified. RESULTS: A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA). CONCLUSIONS: Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification.

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases.

OBJECTIVE: Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. DESIGN: Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1ß. RESULTS: IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. CONCLUSIONS: Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.

Enteric nervous system impairment in gastroschisis.

INTRODUCTION: After surgical repair of gastroschisis, most neonates exhibit severe intestinal dysmotility. We hypothesized that impaired development of the enteric nervous system or interstitial cells of Cajal (ICC) network contributes to impaired intestinal motility in gastroschisis. We evaluated this hypothesis in a rat model of gastroschisis. MATERIAL AND METHODS: Gastroschisis was created surgically in rat fetuses on gestational day 18, under general anesthesia, and small bowel was harvested on day 22. Intestinal weight-to-length (IW/L) ratio, and small-bowel wall thickness were assessed. Specimens were processed for hematoxylin-eosin staining or immunohistochemistry with specific markers for neuronal cells (Hoxb5), glial cells (GFAP, S100), and ICCs (c-kit). Myenteric plexus maturation was assessed morphologically and compared with sham and control fetuses. Stage of development of the myenteric plexus was graded from 1 (mature) to 3 (very immature) comparatively with specimens from E16 to E22 control fetuses. RESULTS: Compared with sham-operated or control fetuses, gastroschisis was associated with increases in mean intestinal weight/intestinal length (IW/L) ratio, and mean thicknesses of the total, muscular, and submucosal layers of the small-bowel wall. The myenteric plexus were present in the small bowel from fetuses with gastroschisis, however all exhibited abnormal myenteric plexus maturation. Thus, of the gastroschisis fetuses, 55% had an aspect similar to the immature myenteric plexus of E19-E20 fetuses and 45% to the very immature mesenteric plexus observed in E16-E18 fetuses. When compared with sham and control groups, ICCs were less abundant in eviscerated small bowel in the gastroschisis group and often exhibited weak c-kit staining or an abnormally round shape without branches. Hoxb5, a marker for enteric neuroblasts and neuronal precursors, was expressed similarly in myenteric plexuses in all groups. S100 or GFAP staining showed the presence of glial cells within the myenteric plexuses in all groups. The S100 expression level was similar in all groups. In contrast, the GFAP expression level was higher in the gastroschisis group than in the sham and control groups. CONCLUSION: Our results suggest that delayed maturation of the enteric nervous system combined with impaired ICC network development may largely explain the intestinal dysmotility seen in neonates with gastroschisis.

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin.

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.

Current smoking differentially affects blood mononuclear cells from patients with Crohn's disease and ulcerative colitis: relevance to its adverse role in the disease.

BACKGROUND: Epidemiologic data suggest that smoking increases the risk and the severity of Crohn's disease (CD), although it may protect patients with ulcerative colitis (UC). To investigate this paradox, we evaluated the effect of cigarette smoke in the function of blood mononuclear cells from healthy subjects and patients with CD or UC in flare up. METHODS: The production of mediators associated with inflammation but also with protective functions was evaluated by enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), following either in vivo or in vitro exposure to cigarette smoke. RESULTS: We found that mononuclear cells from smokers with CD were functionally impaired. These cells secreted lower levels of chemokines and cytokines as compared with nonsmoker counterparts, whereas healthy smokers or smokers with UC were not affected. Similar findings were noted after in vitro exposure to cigarette smoke extract. In addition, cells from patients with CD who smoke presented a defective sensitivity to antiinflammatory or antioxidant protection, and particularly synthesized lower levels of cytoprotective Hsp70. The effects observed were not due to diminished cell viability. Our experiments suggest that cigarette smoke-related responses were largely dependent on oxidative stress generated, and not on the nicotine component. CONCLUSIONS: Overall, our data point out the presence of biological differences between blood mononuclear cells from patients with CD and UC toward cigarette smoke that might support its opposite role in both diseases.

Proof of principle for transfusion of in vitro-generated red blood cells.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34(+) HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 10(10) cRBCs generated under good manufacturing practice conditions and labeled with (51)Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro-generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

Arsenic trioxide reduces 2,4,6-trinitrobenzene sulfonic acid-induced murine colitis via nuclear factor-κB down-regulation and caspase-3 activation.

Arsenic trioxide, As(2)O(3), already used in human anti-cancer therapy, is also an efficient agent against the autoimmune and inflammatory diseases developed in MRL/lpr mice. Inflammatory bowel diseases (IBDs), notably Crohn's disease, which remain without efficient treatment, display autoimmune and inflammatory components. We, therefore, hypothesized that As(2)O( 3) may be active on IBDs. Using the 2,4,6-trinitrobenzene sulfonic acid-induced murine model of colitis, we demonstrate that As(2)O(3) used either in a preventive or a curative mode markedly reduced the induced colitis as assessed by macroscopic and microscopic scores, leading to prolonged mice survival. In addition, As(2)O(3) was able to inhibit NF-κB expression and DNA-binding in colon extracts leading to decreased cytokine gene expression (i.e. tumor necrosis factor-α, interleukin(IL)-1ß, IL-12, IL-17, IL-18, and IL-23). Interestingly, As(2)O(3) also reduced keratinocyte-derived chemokine (KC), inducible nitric oxide synthase (iNOS) mRNA levels, and myeloperoxidase (MPO) protein expression suggesting an impairment of neutrophils. This was associated with a marked increase of procaspase-3 and induced caspase-3 activation. This caspase-3 co-localized with MPO in the remaining neutrophils suggesting that As(2)O( 3) might have eliminated inflamed cells probably by inducing their apoptosis. These results assessed the potent anti-inflammatory effect of As(2)O( 3), that targets both NF-κB and caspase-3 pathways, and suggests a therapeutic potential for Crohn's disease and other severe IBDs.

Regulation of colon cancer cell proliferation and migration by MD-2 activity.

Evidence suggests that signalling through lipopolysaccharide (LPS) has a significant role in the development of gastrointestinal malignancies. We previously demonstrated the critical role of myeloid differentiation (MD)-2, the essential co-receptor of LPS, for induction of cyclooxygenase (Cox)-2 in intestinal epithelial cells. Cyclooxigenase-2 was suggested to play a key role in colorectal cancer through the effects of prostaglandin (PG) E(2) generated. We, therefore, addressed the role of MD-2 in several parameters related to malignancy, namely cell proliferation and migration, using colon cancer cells (HT-29). We found that overexpression of MD-2 confers a significantly greater proliferation and migration capacity to these cells. MD-2-dependent proliferation and migration appeared independent of Cox-2 activity but was reduced by endothelial growth factor receptor (EGFR) neutralizing antibodies as well as by pharmacological inhibition of EGFR tyrosine phosphorylation. We propose that MD-2 overexpression contributes to tumour aggressiveness via a Cox-2-independent excessive EGFR signalling. Moreover, MD-2 expression levels were higher in tissue from patients with colorectal cancer as compared with paired control colorectal mucosa. Our data attest to a role of MD-2 activity in colon cancer epithelial cell proliferation and migration, which may be important in the general correlation between innate immune response, chronic inflammation, and cancer.

Sera from patients with Crohn's disease break bacterial lipopolysaccharide tolerance of human intestinal epithelial cells via MD-2 activity.

Myeloid differentiation (MD)-2 is linked to the cell surface as a Toll-like receptor (TLR) 4-bound protein though may also function as a soluble receptor to enable the lipopolysaccharide (LPS)-driven response. We recently demonstrated the importance of MD-2 either as a cell-associated or as a soluble receptor in the control of intestinal epithelial cell response toward LPS. High levels of circulating MD-2 were recently proposed as a risk factor for infectious/ inflammatory diseases as septic shock. We hypothesized that MD-2 might be present in sera from patients with inflammatory bowel disease and have pathogenic consequences. We analysed MD-2 activity in sera from patients with inflammatory bowel disease or from healthy subjects. We measured MD-2 activity as the capacity to mediate LPS-driven stimulation of intestinal epithelial cells (HT29). We found that sera from patients with inflammatory bowel disease, particularly Crohn's disease, endowed HT29 cells with a markedly higher LPS-dependent stimulating capacity as compared to sera from healthy subjects. The effect of sera was specific for LPS activation and was reduced in the presence of anti-MD-2, and anti-TLR4 antibodies. We conclude that sera from patients with inflammatory bowel disease might contain increased MD-2. This might result in higher local availability of the protein leading to a loss of tolerance toward gut microbiota.

Distinct behaviour of the homeodomain derived cell penetrating peptide penetratin in interaction with different phospholipids.

BACKGROUND: Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and ß-sheet contributions. CONCLUSIONS/SIGNIFICANCE: These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.

Basolateral internalization of GPI-anchored proteins occurs via a clathrin-independent flotillin-dependent pathway in polarized hepatic cells.

In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2-positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.

Galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells.

We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients.

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-kappaB activity, F. prausnitzii had no effect on IL-1beta-induced NF-kappaB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-gamma production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-kappaB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.

The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains.

BACKGROUND: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. CONCLUSIONS/SIGNIFICANCE: The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that the membrane targets for these molecules are preferentially the fluid membrane domains and that the mechanism involves the induction of membrane negative curvature. Consequences on cellular uptake are discussed.

MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells.

Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.

Non-metabolic membrane tubulation and permeability induced by bioactive peptides.

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated "physical endocytosis," which represents a new pathway for peptide cellular internalization.

Cytoplasmic phospholipase A2 expression in human colon adenocarcinoma is correlated with cyclooxygenase-2 expression and contributes to prostaglandin E2 production.

Cytoplasmic phospholipase A2 (cPLA2) has a key role in prostaglandin production. The role of cPLA2 in intestinal tumorigenesis has been suggested, however, contradictory data are found in the literature. We evaluated cPLA2 and cyclooxygenase-2 (COX-2) protein expression in 65 colon carcinomas by immunohistochemistry, and in eight colorectal cancer cell lines by Western Blot. PGE2 production was evaluated by enzyme-immunoassay in the cell lines. We demonstrate that cPLA2 is overexpressed in approximately 50% of colon cancers and cell lines. cPLA2 expression is correlated with COX-2 expression. Both cPLA2 and COX-2 expressions are important in regard to PGE2 production. Our data suggest that cPLA2 might be involved in colon tumor development.

[Transduction peptides: structural-functional analyses in model membranes]. / Peptides de transduction: analyse des relations "structure-fonction" à l'aide de membranes modèles.

Peptide-membrane interaction is the first step required for peptide cell internalization. In this paper we studied the interactions of substance P, Penetratin and an amphiphilic 16mer (RL16) peptide in two different model membranes, giant unilamellar vesicles and large unilamellar vesicles. Penetratin was able to induce the formation of tubes inside the giant vesicles without changes in membrane permeability. On the contrary, RL16 induced the disruption of giant vesicles and the permeabilization of large vesicles. Substance P showed none of these effects.

Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex.

Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with cubilin. By expression in transfected polarized MDCK cells of AMN and several cubilin fragments, including a functional "mini" version of cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with cubilin and co-localized with cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the cubilin-AMN complex to the apical membrane: AMN interacts with the EGF domains of cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.