TNFR1-induced lethal inflammation is mediated by goblet and Paneth cell dysfunction.

Tumor necrosis factor (TNF) is a powerful activator of the immune system and a well-validated target for treatment of autoimmune diseases. Injection of TNF induces systemic lethal inflammation characterized by hypothermia, induction of multiple cytokines, and extensive damage to multiple organs. Previously, we reported that TNF-induced lethal inflammation is strictly TNFR1(P55)-dependent. We also uncovered a crucial role for P55 expression levels in intestinal epithelial cells (IECs), in which P55+/+ expression is sufficient to sensitize to TNF lethality in an otherwise fully protected P55+/- background. Here, we investigated the molecular mechanism that drives TNF toxicity in IECs. Unexpectedly, we found that the degree of TNF-induced enterocyte damage and apoptosis in IECs is equally strong in TNF-sensitive P55+/+ mice and TNF-resistant P55+/- mice. Our results suggest that P55+/+-induced signaling causes goblet and Paneth cell dysfunction, leading to severe epithelial barrier dysfunction. As a result, intestinal permeability and systemic bacterial spread are induced, causing lethal systemic inflammation. In conclusion, we identified P55-induced goblet and Paneth cell dysfunction as a crucial mechanism for TNF-induced systemic and lethal inflammation.

Tissue distribution of the murine phosphomannomutases Pmm1 and Pmm2 during brain development.

The most common type of the congenital disorders of glycosylation, CDG-Ia, is caused by mutations in the human PMM2 gene, reducing phosphomannomutase (PMM) activity. The PMM2 mutations mainly lead to neurological symptoms, while other tissues are only variably affected. Another phosphomannomutase, PMM1, is present at high levels in the brain. This raises the question why PMM1 does not compensate for the reduced PMM2 activity during CDG-Ia pathogenesis. We compared the expression profile of the murine Pmm1 and Pmm2 mRNA and protein in prenatal and postnatal mouse brain at the histological level. We observed a considerable expression of both Pmms in different regions of the embryonic and adult mouse brain. Surprisingly, the expression patterns were largely overlapping. This data indicates that expression differences on the cellular and tissue level are an unlikely explanation for the absence of functional compensation. These results suggest that Pmm1 in vivo does not exert the phosphomannomutase-like activity seen in biochemical assays, but either acts on as yet unidentified specific substrates or fulfils entirely different functions.

Evidence for a cysteine-histidine thioether bridge in functional units of molluscan haemocyanins and location of the disulfide bridges in functional units d and g of the betaC-haemocyanin of Helix pomatia.

In functional units d and g from the betaC-haemocyanin of the gastropod Helix pomatia, aminoacid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units. The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatia d and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cysteine-histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2-thiolhistidine. A peptide with a cysteine-histidine thioether bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris. A cysteine-histidine thioether bridge, which was reported previously for tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.

Further approaches to the quaternary structure of octopus hemocyanin: a model based on immunoelectron microscopy and image processing.

The direction of the polypeptide chains and the location of the functional units in Octopus vulgaris hemocyanin were studied by various methods. Monoclonal antibodies specific for the Ovc (clone Ov409) and Ovg (clone Ov315) functional units produced immunocomplex strings which were examined in the electron microscope. In both cases the immunocomplexes contained more than two hemocyanin molecules in their side view, demonstrating that in the whole hemocyanin neighboring polypeptide chains run in antiparallel directions. The interhemocyanin distances in the immunocomplexes also indicated that Ovg is located inside the cylinder, while Ovc is located in the external layers of functional units. In addition, the fact that the binding point of the Fab arm to the hemocyanin molecule was occasionally visible confirmed the external location of functional unit Ovc. Image processing of the whole hemocyanin cross-linked with dimethyl suberimidate showed that the end-on view is not a perfect cylinder but a regular pentahedron and that the five-arch collar is probably composed of five pairs of functional unit Ovg located inside the cylinder. The accessibility of cross-linked hemocyanin to functional unit-specific polyclonal antibodies, studied in immunoelectrophoresis, showed that Ovb and Ove are highly accessible, while Ovd, Ovf, and Ovg are not. The low accessibility of Ovd may be at least partially explained by its high sugar content which could hamper the accessibility of the antibody to the antigen.