To understand the mechanism by which individual DNA molecules enter nanometer-scale pores, we studied the concentration and voltage dependence of polynucleotide-induced ionic-current blockades of a single alpha-hemolysin ion channel. We find that the blockade frequency is proportional to the polymer concentration, that it increases exponentially with the applied potential, and that DNA enters the pore more readily through the entrance that has the larger vestibule. We also measure the minimum value of the electrical potential that confines a modified polymer inside the pore against random diffusion and repulsive forces.

Similar to other A-B binary toxins, the putative mechanism of anthrax toxin-induced toxinemia suggests that the B-protein, protective antigen (PA), 2 binds to a cell surface receptor (2, 3) and is subsequently cleaved at the N terminus into two fragments, PA 20 and PA 63 (4, 5). Following cleavage, PA 63 oligomerizes into a heptameric prepore on the cell membrane (6). The A-proteins, lethal factor (LF) and edema factor (EF), competitively bind to the heptameric PA 63 , forming the (PA 63 ) 7 -LF/EF complex which is endocytosed (7). Upon acidification of the endosome, the heptameric PA 63 is thought to undergo a conformational change, insert into the membrane, and form a functional transmembrane pore. In this model, LF and EF are subsequently translocated across the endosomal membrane into the cytosol (8). Determining the mechanism of PA 63 -mediated translocation of EF/LF into the cytosol remains an interesting challenge. Here we present proof-of principle for a potential biosensor in anthrax diagnostics and therapeutics. EXPERIMENTAL PROCEDURESProteins-Purified PA 63 , LF, and EF proteins (List Biological Laboratories Inc.) from Bacillus anthracis were used for these studies.Antibody-Hybridoma PA 63 1G3-1-1 was prepared by fusing spleen cells from female BALB/c mice injected with PA 63 with SP2/O-Ag 14 myeloma cells and subcloning positive hybridomas twice by limiting dilution (9, 10). Ascites was produced in BALB/c female mice and was clarified by centrifugation. This ascites monoclonal antibody, 1G3-1-1, prevents binding of LF to PA 63 through steric hindrance (9).Electrophysiology-Channel recordings were carried out with planar lipid bilayer membranes as previously described (11). Briefly, solventfree diphytanoyl phosphatidylcholine membranes were formed on a 50 -100-m diameter hole in a thin Teflon partition. The partition separated two identical Teflon chambers that each contained ϳ2 ml of aqueous solution (0.1 M KCl, 5 mM MES, pH 6.6). Voltage was applied across the membrane via Ag-AgCl electrodes. The current was amplified using a patch-clamp instrument (Axon Instruments 200B), recorded with an analog to digital converter (Axon Instruments DigiData 1322), and analyzed off-line. A negative transmembrane potential drove anions from the cis to the trans chamber. Details of this particular method were summarized elsewhere (12).Channels were formed by adding small aliquots (ϳ100 ng) of the purified PA 63 to the aqueous electrolyte solution bathing one side of the membrane (herein called cis). The formation of individual channels was indicated by the stepwise increases in ionic current monitored at ϩ50 mV applied potential. During recording of the ionic current in the steady-state experiments, the membrane potential was held constant at either ϩ50 or ϩ100 mV. To determine the instantaneous current-voltage (I-V) relationships, we generated current by applying brief (0.5 s) voltage pulses (typically ϩ200 to Ϫ200 mV in 10-mV steps) across the membrane and averaged the first 100 ms of the signal to obtain the inst...

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF TM mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD41 cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4 1 cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. Key termsquantitative multiparameter flow cytometry; CyTOF TM mass cytometry; antibody bound per cell; cryopreserved peripheral blood mononuclear cells; whole blood; lyophilized peripheral blood mononuclear cell; Cyto-Trol; CD4 expression; cell membrane intactness; cell diameter; fixation effect

The current-voltage relationship of a single Staphylococcus aureus alpha-hemolysin (alphaHL) channel is nonlinear, rectifying, and depends on the bulk pH and the ionic strength. The data are described qualitatively by a simple one-dimensional Nernst-Planck analysis in which the fixed charges inside and near the pore's entrances affect the transport of ions through the channel. The distances of these fixed charges from one of the channel's entrances are obtained from the channel's crystal structure. The model demonstrates that rectification of monovalent ion flow through the alphaHL channel can be related to the asymmetry in the location of the ionizable amino acid side chains.

This paper presents material which is intended to assist researchers in identifying and controlling a range of biological, electrical, and other physical parameters that can affect the outcome of in vitro studies with extremely low frequency (ELF) magnetic and electric fields. Brief descriptions of power line magnetic and electric fields are provided and methods for the generation of 60 Hz as well as other ELF fields in the laboratory are surveyed. Methods for calculating and measuring exposure parameters in culture medium are also described. Relating in vitro and internal in vivo exposure conditions across different animal species is discussed to aid researchers in selecting levels of field exposure. The text is purposely elementary, and sometimes brief, with references provided to aid the interested reader in obtaining a fuller understanding of the many topics. Because the range of experimental parameters that can influence the outcome of in vitro studies with ELF fields is so broad, a multidisciplinary approach is normally required to carry out the research.