Tetanus toxin potently stimulates tissue transglutaminase. A possible mechanism of neurotoxicity.

The observation that tetanus toxin (TT) contains two sequences that show homology to known transglutaminase (TGase) substrate sites suggested that the toxin and TGase might interact. This prediction was confirmed by two pieces of evidence. First, TT potently stimulated the enzymatic activity of TGase. The effect was maximal at physiological (micromolar) concentrations of the endogenous TGase regulators calcium and GTP. Second, TT and TGase displayed marked variations of their intrinsic fluorescence properties when they were coincubated, indicating the occurrence of binding between them. TT-TGase binding and TGase activation occurred at similar concentrations of TT and are probably causally related. The activation of TGase, an enzyme present in nerve endings that, when activated, can irreversibly cross-link cellular proteins, might mediate the neurotoxic action of TT.     

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods.
Materials and methods:  Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH).
Results:  In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis /flexispira taxon 8, H. cinaedi , and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH.
Conclusions:  This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.     

Journeys through the Golgi—taking stock in a new era

The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.     

Genetic Loci Involved in Antibody Response to Mycobacterium avium ssp. paratuberculosis in Cattle

Background

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne''s disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision.

Methodology/Principal Findings

The objective of the current study was to localize genes having an impact on Johne''s disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip) to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal.

Conclusion/Significance

The analysis of the genotype data identified several chromosomal regions associated with disease status: a region on chromosome 12 with high significance (P<5×10⁻⁶), while regions on chromosome 9, 11, and 12 had moderate significance (P<5×10⁻⁵). These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information into breeding programmes for the improvement of health status.     

Exiting the Golgi complex

The composition and identity of cell organelles are dictated by the flux of lipids and proteins that they receive and lose through cytosolic exchange and membrane trafficking. The trans-Golgi network (TGN) is a major sorting centre for cell lipids and proteins at the crossroads of the endocytic and exocytic pathways; it has a complex dynamic structure composed of a network of tubular membranes that generate pleiomorphic carriers targeted to different destinations. Live-cell imaging combined with three-dimensional tomography has recently provided the temporal and topographical framework that allows the assembly of the numerous molecular machineries so far implicated in sorting and trafficking at the TGN.     

Morphogenesis of post-Golgi transport carriers

The trans-Golgi network (TGN) is one of the main, if not the main, sorting stations in the process of intracellular protein trafficking. It is therefore of central importance to understand how the key players in the TGN-based sorting and delivery process, the post-Golgi carriers (PGCs), form and function. Over the last few years, modern morphological approaches have generated new insights into the questions of PGC biogenesis, structure and dynamics. Here, we present a view by which the “lifecycle” of a PGC consists of several distinct stages: the formation of TGN tubular export domains (where different cargoes are segregated from each other and from the Golgi enzymes); the docking of these tubular domains onto molecular motors and their extrusion towards the cell periphery along microtubules; the fission of the forming PGC from the donor membrane; and the delivery of the newly formed PGC to its specific acceptor organelle. It is now important to add the many molecular machineries that have been described as operating at the TGN to this “morphofunctional map” of the TGN export process.     

Mechanism of constitutive export from the golgi: bulk flow via the formation,protrusion, and en bloc cleavage of large trans-golgi network tubular domains

Transport of constitutive cargo proteins from the Golgi complex to the plasma membrane (PM) is known to be mediated by large tubular-saccular carriers moving along microtubules. However, the process by which these large structures emerge from the trans-Golgi network (TGN) remains unclear. Here, we address the question of the formation of Golgi-to-PM carriers (GPCs) by using a suitable cluster of morphological techniques, providing an integrated view of their dynamics and three-dimensional structure. Our results indicate that exit from the TGN of a constitutive traffic marker, the VSVG protein, occurs by bulk flow and is a three-step process. First, the formation of a tubular-reticular TGN domain (GPC precursor) that includes PM-directed proteins and excludes other cargo and Golgi-resident proteins. Notably, this step does not require membrane fusion. Second, the docking of this preformed domain on microtubules and its kinesin-mediated extrusion. Finally, the detachment of the extruded domain by membrane fission. The formation of GPCs does not involve cargo concentration and is not associated with the presence of known coat proteins on GPC precursors. In summary, export from the Golgi occurs via the formation, protrusion and en bloc cleavage of specialized TGN tubular-saccular domains.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.