Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.

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ABSTRACT. Objectives. Fibromyalgia has been recently recognized in children and adolescents as juvenile fibromyalgia (JF). In adult fibromyalgia, subjective complaints of nonrestorative sleep and fatigue are supported by altered polysomnographic findings including a primary sleep disorder known as periodic limb movements in sleep (PLMS) in some subjects. Although poor sleep is a diagnostic criterion for JF, few reports in the literature have evaluated specific sleep disturbances. Our objectives were to evaluate in a controlled study the polysomnographic findings of children and adolescents with JF for alterations in sleep architecture as well as possible PLMS not previously noted in this age group.Methods. Sixteen consecutive children and adolescents (15.0 ؎ 2.6 years of age) diagnosed with JF underwent overnight polysomnography. Polysomnography was also performed on 14 controls (14.0 ؎ 2.2 years of age) with no history of an underlying medical condition that could impact on sleep architecture. Respiratory variables, sleep stages, and limb movements were measured during sleep in all subjects.Results. JF subjects differed significantly from controls in sleep architecture. JF subjects presented with prolonged sleep latency, shortened total sleep time, decreased sleep efficiency, and increased wakefulness during sleep. In addition, JF subjects exhibited excessive movement activity during sleep. Six of the JF subjects (38%) were noted to have an abnormally elevated PLMS index (>5/hour), indicating PLMS in these subjects.Conclusion. Our study demonstrated abnormalities in sleep architecture in children with JF. We also noted PLMS in a significant number of subjects. This has not been reported previously in children with this disorder. We recommend that children who are evaluated for JF undergo polysomnography including PLMS assessment. Pediatrics 2000;106(5). URL: http://www.pediatrics.org/ cgi/content/full/106/5/e70; juvenile fibromyalgia; periodic limb movement in sleep; restless legs syndrome.

Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer.

NA MEASUREMENTS AND RESULTS: REM density and the duration of REM cycles increased over the course of the night until the fifth REM cycle, and then stabilized. The duty cycle increased across the first 6 REM cycles. However, the apnea hypopnea index (AHI) did not increase across REM cycles, and was not affected by the changes in REM density or duty cycle. We speculate that the increase in the duty cycle is a compensatory response to increased upper airway loads during sleep, and that this may lead to ventilatory or upper airway muscle fatigue.