Anthrax toxin, a major virulence factor of Bacillus anthracis, gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8-and CMG2-null mice and TEM8/ CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.edema toxin ͉ lethal toxin ͉ tumor endothelium marker-8 B acillus anthracis is a Gram-positive, rod-shaped, sporeforming bacterium, and the causative agent of anthrax. Anthrax toxin is the major virulence factor for this organism and responsible for its lethal effects in the host. Although treatment with antibiotics to eliminate the bacteria can be life-saving at the earlier stages of anthrax disease, once enough toxin has been produced, the disease is often lethal despite treatment. Thus, countermeasures that block toxin or limit its effects are essential at later stages of disease (1). Therefore, a detailed understanding of the interaction between anthrax toxin and the host is needed as a basis for developing improved interventions.Anthrax toxin is a 3-part toxin consisting of protective antigen (PA, 83 kDa), edema factor (EF, 90 kDa), and lethal factor (LF, 89 kDa) (2-4). These 3 proteins are individually nontoxic, but can assemble on the cell surface to form toxic complexes. To intoxicate host mammalian cells, PA binds to its cellular receptors, tumor endothelium marker-8 [TEM8, also named anthrax toxin receptor 1 (ANTXR1)] and capillary morphogenesis protein-2 [CMG2, also named anthrax toxin receptor 2 (ANTXR2)] (5, 6) with the involvement of a coreceptor LRP6 (7,8) and is then proteolytically processed to the active form, cell-surface bound PA63. PA63 spontaneously oligomerizes to form a heptamer that binds and delivers LF and EF into the cytosol. EF, which combines with PA to form edema toxin (ET), is a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP levels, thereby causing diverse effects including impairment of phagocytosis and death of experimental animals (9, 10). LF, which combines with PA to form lethal toxin (LT), is a zinc-dependent metalloproteinase that cleaves and inactivates the mitogen-activated protein kinase kinases (MEKs) 1-4, 6, and 7 (11-13), blocking the ERK, p38, and Jun...

On the basis of the strategy of promoting thermostability of α-diimine nickel catalyst by ligand backbone framework, a series of α-diimine nickel(II) complexes with bulky camphyl or diaryl-substituted backbones, [2,6-(R2)2C6H3−NC(R1)−C(R1)N−2,6-(R2)2C6H3]NiBr2 (1a, R1 = Ph, R2 = CH3; 2a, R1 = 4-methylphenyl, R2 = CH3; 3a, R1 = 4-fluorophenyl, R2 = CH3; 4a, R1 = camphyl, R2 = CH3; 4b, R1 = camphyl, R2 = i-Pr), were synthesized and used as catalyst precursors for ethylene polymerization. Crystallographic analysis revealed that the bulky camphyl backbone has a valid steric-effect on the nickel center by blocking the axial site for the metal center and suppressing the potential rotation of the CAr−N bond. Ethylene polymerizations catalyzed by these nickel α-diimine complexes activated by MAO were systematically investigated and the influences of the substituted backbones as well as reaction temperature on the catalytic activity, molecular weight and branching structure of the polymers were evaluated. It was found that the catalysts containing a camphyl backbone have excellent thermal stability and polymer structure control for ethylene polymerizations. Even at 80 °C, the 4b/MAO system still kept high activity and relatively stable kinetics and produced high molecular weight polyethylene. Moreover, the branching degrees and branched chain distribution of the polyethylenes obtained by the complex could also be controlled by tuning the reaction temperature.

SummaryThe current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of D -alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of L -alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

On the basis of different approaches for modifying α-diimine palladium catalysts, a series of methyl chloride palladium complexes with various α-diimine ligand backbones were synthesized and characterized. The corresponding cationic palladium complex chelating esters were further obtained by treatment of methyl chloride palladium complexes with methyl acrylate (MA). It was observed that decomposition of a cationic palladium complex chelating ester can occur to produce a new cationic palladium complex chelating two ligands and two counteranions, which provides a new pathway for deactivation of palladium catalysts and formation of palladium black by a fragmentation pattern with ester loss. These α-diimine palladium catalysts were employed in the homopolymerization of ethylene and copolymerization of ethylene and MA to evaluate substituent effects of the ligand backbone. A bulky camphyl α-diimine palladium catalyst was found to show better thermal stability and afford high-molecular-weight copolymer with higher incorporation of polar monomer. Longstanding living polymerization of ethylene was also achieved within 12 h using a bulky camphyl α-diimine palladium catalyst.

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptormediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and͞or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid ␣-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.