Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties.

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.     

Galanin-like immunoreactivity in extrinsic and intrinsic nerves to the gut of the Atlantic cod,Gadus morhua,and the effect of galanin on the smooth muscle of the gut

The presence of galanin-like immunoreactivity in nerves to the stomach of the Atlantic cod has been investigated by immunohistochemistry. The distribution of ganglion cells showing galanin-like immunoreactivity was compared with the total distribution in nerves and ganglia. Projection studies were made to determine the origin of the galanin neurons. The effect of galanin was studied in smooth muscle strip preparations of the gut wall and arteries. Galanin-like immunoreactive ganglion cells frequently occurred along the vagal branches to the stomach. Most of them projected cranially. Immunoreactive nerve fibres were present in all layers of the gut and around arterial branches on the surface of the stomach. Ligations of the vagus and splanchnic nerves produced accumulations of immunoreactive material on both sides of the ligature. Galanin produced weak contractile effects unaffected by tetrodotoxin on the gut wall and on gut arteries. It is concluded that a population of the ganglion cells along the vagus nerve in the Atlantic cod contains a galanin-like peptide. Some of these cells may be parts of autonomic parasympathetic pathways innervating the gut of the Atlantic cod, having direct excitatory effects on the smooth muscles of the gut wall and gut arteries.     

Persistent versus transient tree encroachment of temperate peat bogs: effects of climate warming and drought events

Peatlands store approximately 30% of global soil carbon, most in moss‐dominated bogs. Future climatic changes, such as changes in precipitation patterns and warming, are expected to affect peat bog vegetation composition and thereby its long‐term carbon sequestration capacity. Theoretical work suggests that an episode of rapid environmental change is more likely to trigger transitions to alternative ecosystem states than a gradual, but equally large, change in conditions. We used a dynamic vegetation model to explore the impacts of drought events and increased temperature on vegetation composition of temperate peat bogs. We analyzed the consequences of six patterns of summer drought events combined with five temperature scenarios to test whether an open peat bog dominated by moss (Sphagnum) could shift to a tree‐dominated state. Unexpectedly, neither a gradual decrease in the amount of summer precipitation nor the occurrence of a number of extremely dry summers in a row could shift the moss‐dominated peat bog permanently into a tree‐dominated peat bog. The increase in tree biomass during drought events was unable to trigger positive feedbacks that keep the ecosystem in a tree‐dominated state after a return to previous ‘normal’ rainfall conditions. In contrast, temperature increases from 1 °C onward already shifted peat bogs into tree‐dominated ecosystems. In our simulations, drought events facilitated tree establishment, but temperature determined how much tree biomass could develop. Our results suggest that under current climatic conditions, peat bog vegetation is rather resilient to drought events, but very sensitive to temperature increases, indicating that future warming is likely to trigger persistent vegetation shifts.     

Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.