Three-dimensional structure of tropism-switching Bordetella bacteriophage.

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail "hub," six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7A resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.

Passive transfer of IgG anti-GM1 antibodies impairs peripheral nerve repair.

Anti-GM1 antibodies are present in some patients with autoimmune neurological disorders. These antibodies are most frequently associated with acute immune neuropathy called Guillain-Barré syndrome (GBS). Some clinical studies associate the presence of these antibodies with poor recovery in GBS. The patients with incomplete recovery have failure of nerve repair, particularly axon regeneration. Our previous work indicates that monoclonal antibodies can inhibit axon regeneration by engaging cell surface gangliosides (Lehmann et al., 2007). We asked whether passive transfer of human anti-GM1 antibodies from patients with GBS modulate axon regeneration in an animal model. Human anti-GM1 antibodies were compared with other GM1 ligands, cholera toxin B subunit and a monoclonal anti-GM1 antibody. Our results show that patient derived anti-GM1 antibodies and cholera toxin beta subunit impair axon regeneration/repair after PNS injury in mice. Comparative studies indicated that the antibody/ligand-mediated inhibition of axon regeneration is dependent on antibody/ligand characteristics such as affinity-avidity and fine specificity. These data indicate that circulating immune effectors such as human autoantibodies, which are exogenous to the nervous system, can modulate axon regeneration/nerve repair in autoimmune neurological disorders such as GBS.

Isolation, characterization and transcriptional regulation of {\it Xenopus myod\/}

I have cloned cDNAs corresponding to two distinct genes, Xlmf1 and Xlmf25, which encode skeletal muscle-specific, transcriptional regulatory proteins. These proteins are members of the helix-loop-helix family of DNA binding factors, and are most homologous to MyoD1. These two genes have disparate temporal expression patterns during early embryogenesis; although, both transcripts are present exclusively in skeletal muscle of the adult. Xlmf1 is first detected 7 hours after fertilization, shortly after the midblastula transition. Xlmf25 is detected in maternal stores of mRNA, during early cleavage stages of the embryo and throughout later development. Both Xlmf1 and Xlmf25 transcripts are detected prior to the expression of other, previously characterized, muscle-specific genes. The ability of Xlmf1 and Xlmf25 to convert mouse 10T1/2 fibroblasts to a myogenic phenotype demonstrates their activity as myogenic regulatory factors. Additionally, Xlmf1 and Xlmf25 can directly transactivate a reporter gene linked to the muscle-specific, muscle creatine kinase (MCK) enhancer. The functional properties of Xlmf1 and Xlmf25 proteins were further explored by investigating their interactions with the binding site in the MCK enhancer. Analysis of dissociation rates revealed that Xlmf25-E12 dimers had a two-fold lower avidity for this site than did Xlmf1-E12 dimers. Clones containing genomic sequence of Xlmf1 and Xlmf25 have been isolated. Reporter gene constructs containing a lac-z gene driven by Xlmf1 regulatory sequences were analyzed by embryo injections and transfections into cultured muscle cells. Elements within $-$200 bp of the transcription start site can promote high levels of muscle specific expression. Embryo injections show that 3500 bp of upstream sequence is sufficient to drive somite specific expression. EMSAs and DNAse I footprint analysis has shown the discrete interaction of factors with several cis-elements within 200 bp of the transcription start site. Mutation of several of these elements shows a positive requirement for two CCAAT boxes and two E boxes. It is evident from the work performed with this promoter that Xlmf1 is tightly regulated during muscle cell differentiation. This is not surprising given the fact that its gene product is crucial to the determination of cell fate choices. ^

Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

DETERMINANTS OF THE SELECTIVE PHAGOCYTOSIS OF CARCINOGENIC PARTICULATE NICKEL COMPOUNDS

Certain inorganic nickel compounds such as crystalline NiS and Ni(,3)S(,2) are potent inducers of carcinogenesis and in vitro cell transformation, while several closely-related compounds such as amorphous NiS are essentially devoid of genotoxic activity. The phenomenon of selectivity of phagocytosis among such particulate nickel compounds has been hypothesized to account for their widely varying toxicological potency, yet the determinants of this selectivity have not been well characterized. Extracellular medium composition, particle dissolution, and particle surface charge were examined as potential determinants of selective phagocytosis for the carcinogenic crystalline and noncarcinogenic amorphous modifications of NiS. Selectivity and avidity of uptake of crystalline NiS by CHO cells was not dependent upon serum: phagocytosis of crystalline, but not amorphous NiS proceeded readily in a minimal salts/glucose medium at 37(DEGREES)C. The evolution of phagocytosis-inhibiting Ni(II) from the surface of amorphous NiS particles did not demonstrably contribute to the lower uptake of these noncarcinogenic particles despite their somewhat greater dissolution rate than the readily phagocytosed crystalline NiS particles. Significant differences in surface charge were noted between crystalline and amorphous NiS, the former being more negative in charge in distilled water suspension. Exposure of amorphous NiS particles to the vigorously reducing environment of a LiAlH(,4) solution under an inert atmosphere resulted in the particles' acquisition of a more negative surface charge. Amorphous NiS particles thus treated were phagocytosed by CHO cells to an extent similar to that of untreated crystalline NiS particles and likewise were shown to induce morphological transformation of primary Syrian hamster embryo cells with a similar potency. The potentiation of uptake characteristic of LiAlH(,4)-treated amorphous NiS was lost gradually upon storage of particles in ambient oxygenated atmosphere and was lost rapidly by apparent particle surface oxidation in aerated distilled water suspensions aged for up to 7 days. Concomitant with this loss of uptake there occurred a loss of negative surface charge. These results suggest the predominant role of particle surface charge rather than adsorbed serum components or particle dissolution as a determinant of selective phagocytosis among particulate nickel compounds. ^