Interplay between bile acid metabolism and microbiota in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) physiopathology is multifactorial and roles for both microbiota and bile acid (BA) modifications have been proposed. We investigated role of dysbiosis, transit pattern and BA metabolism in IBS. METHODS: Clinical data, serum, and stool samples were collected in 15 healthy subjects (HS), 16 diarrhea-predominant (IBS-D) and 15 constipation-predominant IBS (IBS-C). Fecal microbiota composition was analyzed by real-time PCR. Sera and fecal BA profiles, 7α-C4 levels, and in vitro BA transformation activity by fecal microbiota were measured by mass spectrometry. Serum Fibroblast Growth Factor 19 (FGF19) was assayed by ELISA. KEYS RESULTS: Dysbiosis was present in IBS patients with an increase in Escherichia coli in IBS-D patients (p = 0.03), and an increase in Bacteroides (p = 0.01) and Bifidobacterium (p = 0.04) in IBS-C patients. Sera primary and amino-conjugated BA were increased in IBS-D (63.5 ± 5.5%, p = 0.01 and 78.9 ± 6.3%, p = 0.03) and IBS-C patients (55.9 ± 5.5%, p = 0.04 and 65.3 ± 6.5%, p = 0.005) compared to HS (37.0 ± 5.8% and 56.7 ± 8.1%). Serum 7α-C4 and FGF19 levels were not different among all three groups. Fecal primary BA were increased in IBS-D patients compared to HS, including chenodeoxycholic acid which has laxative properties (25.6 ± 8.5% vs 3.5 ± 0.6%, p = 0.005). Bile acid deconjugation activity was decreased in IBS-D (p = 0.0001) and IBS-C (p = 0.003) feces. Abdominal pain was positively correlated with serum (R = 0.635, p < 0.001) and fecal (R = 0.391, p = 0.024) primary BA. CONCLUSIONS & INFERENCES: Different sera and fecal BA profiles in IBS patients could be secondary to dysbiosis and further differences between IBS-C and IBS-D could explain stool patterns. This study opens new fields in IBS physiopathology and suggests that modification of BA profiles could have therapeutic potential.

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease.

BACKGROUND: Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. METHODS: Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. RESULTS: The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. CONCLUSIONS: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Unsaturated acyl chains dramatically enhanced cellular uptake by direct translocation of a minimalist oligo-arginine lipopeptide.

The recurring issue with cell penetrating peptides is how to increase direct translocation vs. endocytosis, to avoid premature degradation. Acylation by a cis unsaturated chain (C22:6) of a short cationic peptide provides a new rational design to favour diffuse cytosolic and dense Golgi localisations.

Rotavirus assembly: an alternative model that utilizes an atypical trafficking pathway.

We review here recent advances in our knowledge on trafficking and assembly of rotavirus and rotaviral proteins in intestinal cells. Assembly of rotavirus has been extensively studied in nonpolarized kidney epithelial MA104 cells, where several data indicate that most if not all the steps of rotavirus assembly take place within the endoplasmic reticulum (ER) and that rotavirus is release upon cell lysis. We focus here on data obtained in intestinal cells that argue for another scheme of rotavirus assembly, where the final steps seem to take place outside the ER with an apically polarized release of rotavirus without significant cell lysis. One of the key observations made by different groups is that VP4 and other structural proteins interact substantially with specialized membrane microdomains enriched in cholesterol and sphingolipids termed rafts. In addition, recent data point to the fact that VP4 does not localize within the ER or the Golgi apparatus in infected intestinal cells. The mechanisms by which VP4, a cytosolic protein, may be targeted to the apical membrane in these cells and assembles with the other structural proteins are discussed. The identification of cellular proteins such as Hsp70, flotillin, rab5, PRA1 and cytoskeletal components that interact with VP4 may help to define an atypical polarized trafficking pathway to the apical membrane of intestinal cells that will be raft-dependent and by-pass the classical exocytic route.

Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

Hypoxia enhances Ecto-5'-Nucleotidase activity and cell surface expression in endothelial cells: role of membrane lipids.

Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5'-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5'-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O2 for 18 hours) induced a 2-fold increase of Ecto-5'-Nucleotidase activity (Vmax 19.78+/-0.53 versus 8.82+/-1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5'-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5'-Nucleotidase endocytosis, without modification of Ecto-5'-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5'-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5'-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5'-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5'-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.

Lovastatin enhances ecto-5'-nucleotidase activity and cell surface expression in endothelial cells: implication of rho-family GTPases.

Extracellular adenosine production by the GPI-anchored Ecto-5'-Nucleotidase (Ecto-5'-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5'Nu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5'-Nu stimulation by HRIs, Ecto-5'-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5'-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5'-Nu and decreased endocytosis of Ecto-5'-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5'-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5'-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5'-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5'-Nu endocytosis through a decrease of Rho-GTPases isoprenylation.

The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts.

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.

Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis.

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

Human mesothelial cells express tissue factor when switched to proliferating state. Pharmacological modulation in vitro.

Covering the inner surface of small-diameter arterial prostheses with endothelial cells (ECs) has been proposed as a means of improving biocompatibility and thrombosis resistance. Because the availability of autologous ECs is limited, autologous human mesothelial cells (HMCs) have been suggested as a substitute for ECs. However, HMCs express tissue factor (TF) in vitro, a deleterious characteristic in vivo. We investigated the distribution of TF antigen and of its inhibitor, tissue factor pathway inhibitor, on HMCs and the effect of pharmacological agents on TF expression. TF antigen was measured by enzyme-linked immunosorbent assay and localized by confocal microscopy. Three distinct pools of TF antigen were demonstrated: within the cells, at the cell surface, and in the extracellular matrix. The effects of ilomedin (10 microg/ml) and heparin (500 U/ml), known to affect procoagulant activity, were evaluated by incubating HMCs for 24 h with or without these agents. Ilomedin, but not heparin, decreased TF antigen expression by 30% (P < 0.05). Despite the theoretical potential of HMCs as a vascular prosthesis lining, TF expression by HMCs remains a major drawback. A technique capable of blocking TF expression until the HMCs return to their resting state is needed. Genetic manipulation of HMCs may hold promise for such a technique.

Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells.

Oxygen (O(2)) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O(2)). We observed a rapid and reversible growth arrest of the cells after only 24 h of O(2) exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells.

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.

Differential effect of GalNAcalpha-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern.

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.

A heat labile soluble factor from Bacteroides thetaiotaomicron VPI-5482 specifically increases the galactosylation pattern of HT29-MTX cells.

The aim of this work was to set up and validate an in vitro model to study a molecular response of an intestinal host cell line (HT29-MTX), to a non-pathogen microflora component. We found that Bacteroides thetaiotaomicron strain VPI-5482 had the capacity to change a specific glycosylation process in HT29-MTX cells via a mechanism that involved a soluble factor. Differentiated HT29-MTX cells were grown in the presence of 20% of spent culture supernatant from the B. thetaiotaomicron during 10 days. Glycosylation processes were followed using a large panel of lectins and analysed using confocal microscopy, western blotting and flow cytometry techniques. Our results show that a B. thetaiotaomicron soluble factor modified specifically the galactosylation pattern of HT29-MTX cells, whereas other glycosylation steps remained mainly unaffected. Further characterization of this soluble factor indicates that it is a heat labile, low molecular weight compound. Reverse transcript-PCR (RT-PCR) analysis was unable to show any significant change in mRNA expression level of the main galactosyltransferases expressed in HT29-MTX cells. By contrast, galactosyltransferase activities dramatically increased in HT29-MTX cells treated by the soluble extract of B. thetaiotaomicron, suggesting a post-translational regulation of these activities. Our in vitro model allowed us to study the cross-talk between a single bacteria and intestinal cells. The galactosylation process appears to be a target of this communication, thus uncovering a new window to study the functional consequences of co-operative symbiotic bacterial-host interactions.

The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif.

Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-Phi (where Phi is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-Phi motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280-288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomal-sorting motif, the core of which was delineated to YFPQA (amino acids 281-285).

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

Apical secretion and sialylation of soluble dipeptidyl peptidase IV are two related events.

The role of glycans in the apical targeting of proteins in epithelial cells remains a debated question. We have expressed the mouse soluble dipeptidyl peptidase IV (DPP IV ectodomain) in kidney (MDCK) and in intestinal (Caco-2) epithelial cell lines, as a model to study the role of glycosylation in apical targeting. The mouse DPP IV ectodomain was secreted mainly into the apical medium by MDCK cells. Exposure of MDCK cells to GalNac-alpha-O-benzyl, a drug previously described as an inhibitor of mucin O-glycosylation, produced a protein with a lower molecular weight. In addition this treatment resulted in a decreased apical secretion and an increased basolateral secretion of mouse DPP IV ectodomain. When expressed in Caco-2 cells, the mouse DPP IV ectodomain was secreted mainly into the basolateral medium. However, BGN was still able to decrease the amount of apically secreted protein and to increase its basolateral secretion. Neuraminidase digestion showed that the most striking effect of BGN was a blockade of DPP IV sialylation in both MDCK and Caco-2 cells. These results indicate that a specific glycosylation step, namely, sialylation, plays a key role in the control of the apical targeting of a secreted DPP IV both in MDCK and Caco-2 cells.

Rotavirus spike protein VP4 is present at the plasma membrane and is associated with microtubules in infected cells.

VP4 is an unglycosylated protein of the outer layer of the capsid of rotavirus. It forms spikes that project from the outer layer of mature virions, which is mainly constituted by glycoprotein VP7. VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. Previous studies indicated that VP4 is located in the space between the periphery of the viroplasm and the outside of the endoplasmic reticulum in rotavirus-infected cells. Confocal microscopy of infected MA104 monolayers, immunostained with specific monoclonal antibodies, revealed that a significant fraction of VP4 was present at the plasma membrane early after infection. Another fraction of VP4 is cytoplasmic and colocalizes with beta-tubulin. Flow cytometry analysis confirmed that at the early stage of viral infection, VP4 was present on the plasma membrane and that its N-terminal region, the VP8* subunit, was accessible to antibodies. Biotin labeling of the infected cell surface monolayer with a cell-impermeable reagent allowed the identification of the noncleaved form of VP4 that was associated with the glycoprotein VP7. The localization of VP4 was not modified in cells transfected with a plasmid allowing the expression of a fusion protein consisting of VP4 and the green fluorescent protein. The present data suggest that VP4 reaches the plasma membrane through the microtubule network and that other viral proteins are dispensable for its targeting and transport.

Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells.

The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells.

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.