Claudins create charge-selective channels in the paracellular pathway between epithelial cells

Epithelia separate tissuespaces by regulating the passage of ions, solutes, and water throughboth the transcellular and paracellular pathways. Paracellularpermeability is defined by intercellular tight junctions, which varywidely among tissues with respect to solute flux, electricalresistance, and ionic charge selectivity. To test the hypothesis thatmembers of the claudin family of tight junction proteins create chargeselectivity, we assessed the effect of reversing the charge of selectedextracellular amino acids in two claudins using site-directedmutagenesis. Claudins were expressed in cultured Madin-Darby caninekidney cell monolayers under an inducible promoter, and clones werecompared with and without induction for transmonolayer electricalresistance and dilution potentials. Expression and localization ofclaudins were determined by immunoblotting, immunofluorescencemicroscopy, and freeze-fracture electron microscopy. We observed thatsubstituting a negative for a positive charge at position 65 in thefirst extracellular domain of claudin-4 increased paracellularNa⁺ permeability. Conversely, substituting positive fornegative charges at three positions in the first extracellular domainof claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na⁺ to Cl.These results support a model where claudins create charge-selective channels in the paracellular space.

     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

A first record of Phlebotominae from Argentinean Patagonia (Diptera: Psychodidae: Phlebotominae)

A species of the Lutzomyia oswaldoi group is recorded from the Valcheta stream basin, Somuncura plateau, Patagonia. It represents the World southermost record of a species of Phlebotominae, apparently supporting the hypothesis about the relictual character of several components of the Somuncura plateau biota, particularly for those species belonging to the Paranaense lineages.     

Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.     

Mass-spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N- and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS.     

Genetic variation in three species of Hordeum,and the selection of accessions for the Barley Core Collection

Starch gel electrophoresis of isozymes was used to investigate the level and distribution of genetic variation in accessions of three wild, diploid Hordeum species. Allelic variation was assessed for seven isozyme loci and used in the statistical analysis of 18 accessions of Hordeum brachyantherum subsp. californicum, 29 accessions of H. murinum subsp. glaucum and 11 accessions of H. pusillum. A major purpose with this study was to evaluate the selection of accessions for the international Barley Core Collection, complementing existing passport data. Allelic variation was found in all three species. The variation was mainly between accessions, as most accessions were fixed for certain alleles. Since all three species are self-pollinated, this was anticipated. The fact that the accessions have undergone one or several regeneration cycles from a rather limited number of individuals, after being collected, likely also have reduced the variation. The accessions were clustered by subjecting the genetic distances between them to UPGMA statistical treatment. The clusters in the dendrograms displayed for H. brachyantherum subsp. californicum and H. pusillum distinct patterns related to the geographical origin of the accessions. The accessions of H. murinum subsp. glaucum were less variable than the other two, and did not reveal such a clear pattern. By comparing the already made, geographically based, selection of accessions with the results from this study, it was evident that the selection had worked fairly well for H. brachyantherum subsp. californicum, but less so for the other two species.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration

We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca(2+)](i) and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCepsilon to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.