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The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 A resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 A in length, that runs along the sevenfold axis and ranges from 14 A to 46 A in diameter. The lytic, transmembrane domain comprises the lower half of a(More)
We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus alpha-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of(More)
Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into(More)
A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue(More)
The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(DeltaCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit(More)
There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells. We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents,(More)
The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection,(More)
In this study, the charge selectivity of staphylococcal alpha-hemolysin (alphaHL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anion-selective versions of alphaHL, including the wild-type pore and various mutants, become more anion selective when beta-cyclodextrin (betaCD) is lodged within the(More)
Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit(More)