Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (RI and RII) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the RI and RII subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-myristylation and RII subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the RII subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the RI and RII subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the RII subunit, but not the RI subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that RII subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. RII subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.

Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118–140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126–140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β-barrel. In contrast, human granulocytes were found to bind α-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

Synthesis and in vivo murine evaluation of Na4[1-(1′-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy

Reaction of the normal isomer of [B20H18]2− and the protected thiol anion, [SC(O)OC(CH3)3]−, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4− directly and in good yield. The isomer produced under mild conditions is characterized by an apical–apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2− has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional 11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3−, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4−. The sodium salt of the resulting [B20H17SH]4− ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumors in vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 μg of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy.

Capabilities of liposomes for topological transformation

Dynamic behaviors of liposomes caused by interactions between liposomal membranes and surfactant were studied by direct real-time observation by using high-intensity dark-field microscopy. Solubilization of liposomes by surfactants is thought to be a catastrophic event akin to the explosion of soap bubbles in the air; however, the actual process has not been clarified. We studied this process experimentally and found that liposomes exposed to various surfactants exhibited unusual behavior, namely continuous shrinkage accompanied by intermittent quakes, release of encapsulated liposomes, opening up, and inside–out topological inversion.

Evidence that DNA damage triggers interleukin 10 cytokine production in UV-irradiated murine keratinocytes.

UV irradiation interferes with the induction of T cell-mediated immune responses, in part by causing cells in the skin to produce immunoregulatory cytokines. Recent evidence implicates UV-induced DNA damage as a trigger for the cascade of events leading to systemic immune suppression in vivo. However, to date, there has been no direct evidence linking DNA damage and cytokine production in UV-irradiated cells. Here we provide such evidence by showing that treatment of UV-irradiated murine keratinocytes in vitro with liposomal T4 endonuclease V, which accelerates the repair of cyclobutylpyrimidine dimers in these cells, inhibits their production of immunosuppressive cytokines, including interleukin 10. Application of these liposomes to murine skin in vivo also reduced the induction of interleukin 10 by UV irradiation, whereas liposomes containing heat-inactivated T4 endonuclease V were ineffective. These results support our hypothesis that unrepaired DNA damage in the skin activates the production of cytokines that down-regulate immune responses initiated at distant sites.

Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.