A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.
To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.
Despite its benefits as biological insecticide, Bacillus thuringiensis bears enterotoxins, which can be responsible for a diarrhoeal type of food poisoning. Thus, all 24 isolates from foodstuffs, animals, soil and commercially used biopesticides tested in this study showed the genetic prerequisites necessary to provoke the disease. Moreover, though highly strain-specific, various isolates were able to germinate and also to actively move, which are further requirements for the onset of the disease. Most importantly, all isolates could grow under simulated intestinal conditions and produce significant amounts of enterotoxins. Cytotoxicity assays classified 14 isolates as highly, eight as medium and only two as low toxic. Additionally, growth inhibition by essential oils (EOs) was investigated as preventive measure against putatively enteropathogenic B. thuringiensis. Cinnamon Chinese cassia showed the highest antimicrobial activity, followed by citral, oregano and winter savory. In all tests, high strain-specific variations appeared and must be taken into account when evaluating the hazardous potential of B. thuringiensis and using EOs as antimicrobials. Altogether, the present study shows a non-negligible pathogenic potential of B. thuringiensis, independently from the origin of isolation. Generally, biopesticide strains were indistinguishable from other isolates. Thus, the use of these pesticides might indeed increase the risk for consumers’ health. Until complete information about the safety of the applied strains and formulations is available, consumers or manufacturers might benefit from the antimicrobial activity of EOs to reduce the level of contamination.
Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.
We have isolated and characterized a population of brain macrophages from normal and scrapie-infected mice. The cells are phagocytic, possess Fc-IgG receptors, Mac-1 surface antigen and proliferate in the presence of macrophage colony stimulating factor. They resemble microglia in that they have a plasmalemmal distribution of the enzyme nucleoside diphosphatase, a property tht is characteristic of microglia in situ. In two of the three combinations of scrapie agent and mouse strain examined, the number of brain macrophages was several fold higher than in normal control mice. The increase was not observed in mice infected intraperitoneally or in control mice inoculated with normal brain homogenate. The increase is detectable as early as 3-5 weeks postinoculation. The agent/host combination that failed to show an increase in brain macrophages is one that develops large numbers of amyloid plaques. These observations suggest that these cells are closely associated with the scrapie pathogenic process in the CNS. The failure of these cells to increase in the plaque forming model of scrapie disease also suggests that they play a role in the control of CNS amyloidogenesis.
To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.
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