EFFECTS OF A RESTORED ELK POPULATION ON SOILS, VEGETATION, AND WATER QUALITY IN EASTERN KENTUCKY

A restored elk (Cervus elaphus) population in eastern Kentucky may be affecting ecosystem processes in a landscape composed of reclaimed grassland expanses and isolated forest remnants. Elk routinely select forested ridge-tops as resting and ruminating sites. These locations are characterized by sparse or absent leaf litter, trampled and diminished vegetation, large deposits of dung, and urine-saturated soils. In fall 2003, a series of fenced ungulate exclosures were constructed; 8 on highly disturbed forested ridge-tops and 8 on reclaimed grasslands. Soil analyses measured % moisture, pH, total nitrogen, total carbon, ammonium, nitrate, phosphorus, and major extractable cations in 0-10 cm and 10-20 cm cores. Litter depths and percentages of bare ground, vegetative cover, litter cover, and woody debris were measured. Sediment and water samples were collected monthly from sediment traps on reference and experimental ridge-tops. Studentfs t-tests were used to determine significance (p . 0.10) between treatments. Disturbed ridge-tops had higher soil ammonium (0.68 mg/kg, 10-20 cm) than reference sites (0.25 mg/kg) in spring 2004 and lower ammonium (0.72 mg/kg, 0-10 cm; 0.44 mg/kg, 10-20 cm) than reference sites (1.80 mg/kg, 0-10 cm; 0.94 mg/kg, 10-20 cm) in summer 2004. Total carbon was higher inside (67.57 g/kg, 0-10 cm) than outside (45.38 g/kg) of ridge-top exclosures in fall 2004. Soil moisture, litter depths, and vegetative cover were generally lower, while % bare ground was higher on disturbed ridge-tops. Sediment collected from traps averaged 2.21g/m2 inside exclosures, 2.86 g/m2 outside exclosures, and 0.39 g/m2 on reference ridge-tops. These data suggest that this reintroduced species is changing several attributes of the Cumberland Plateau landscape. The lack of a predator such as the gray wolf (Canis lupis) or cougar (Puma concolor) likely contributes to the development of habitual elk use of landscape features such as remnant ridge-top forests. Such concentrated use may create conditions for the colonization of certain plant species including rare natives and invasive exotics. Continued monitoring of high use areas is recommended so that managers can fully understand the long-term impact of the return of this large, gregarious herbivore, and that appropriate management actions can be developed to promote native biodiversity

Radical Stress Is More Cytotoxic in the Nucleus than in Other Organelles

Contains fulltext : 209354.pdf (publisher's version ) (Open Access

VAMP8-mediated NOX2 recruitment to endosomes is necessary for antigen release

Contains fulltext : 178043.pdf (publisher's version ) (Open Access)Cross-presentation of foreign antigen in major histocompatibility complex (MHC) class I by dendritic cells (DCs) requires activation of the NADPH-oxidase NOX2 complex. We recently showed that NOX2 is recruited to phagosomes by the SNARE protein VAMP8 where NOX2-produced reactive oxygen species (ROS) cause lipid oxidation and membrane disruption, promoting antigen translocation into the cytosol for cross-presentation. In this study, we extend these findings by showing that VAMP8 is also involved in NOX2 trafficking to endosomes. Moreover, we demonstrate in both human and mouse DCs that absence of VAMP8 leads to decreased ROS production, lipid peroxidation and antigen translocation, and that this impairs cross-presentation. In contrast, knockdown of VAMP8 did not affect recruitment of MHC class I and the transporter associated with antigen processing 1 (TAP1) to phagosomes, although surface levels of MHC class I were reduced. Thus, in addition to a secretory role, VAMP8-mediates trafficking of NOX2 to endosomes and phagosomes and this promotes induction of cytolytic T cell immune responses

Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery

Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as beta-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (approximately 50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex

Chromogranin A regulates gut permeability via the antagonistic actions of its proteolytic peptides

AIM: A 'leaky' gut barrier has been implicated in the initiation and progression of a multitude of diseases, e.g., inflammatory bowel disease (IBD), irritable bowel syndrome, and celiac disease. Here we show how pro-hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352-372 ), the most abundant CgA-derived proteolytic peptide, affect the gut barrier. METHODS: Colon tissues from region-specific CST-knockout (CST-KO) mice, CgA-knockout (CgA-KO) and WT mice were analyzed by immunohistochemistry, Western blot, ultrastructural and flowcytometry studies. FITC-dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250-301 ). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. RESULTS: Plasma levels of CST were elevated in IBD patients. CST-KO mice displayed (i) elongated tight, adherens junctions and desmosomes similar to IBD patients, (ii) elevated expression of Claudin 2, and (iii) gut inflammation. Plasma FITC-dextran measurements showed increased intestinal paracellular permeability in the CST-knockout mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST-knockout mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA-knockout mice supplementation with CST and/or PST in CgA-KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. CONCLUSION: The pro-hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST

Hypoxia potentiates monocyte-derived dendritic cells for release of tumor necrosis factor alpha via MAP3K8

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

Contains fulltext : 201259.pdf (publisher's version ) (Open Access

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane