DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA.

The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.

Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation.

Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.

Vinpocetine is a highly potent neuroprotectant against veratridine-induced cell death in primary cultures of rat cerebral cortex.

The effects of vinpocetine and phenytoin against veratridine-induced cell death were investigated in primary cultures of rat cerebral cortex. Toxicity was evaluated by phase contrast microscopy and quantified by measuring lactic dehydrogenase leakage from damaged cells. Vinpocetine was highly potent in inhibiting the cell death evoked by veratridine. The concentrations of the drug evoking 50% protection (IC50 values) against 100 microM (maximal response) and 50 microM (half-maximal response) veratridine were 490 nM and 63 nM, respectively. The protective efficacy of vinpocetine exceeded about 100-fold that of phenytoin (IC50 = 44.2 microM against 100 microM veratridine), a prototype sodium-channel blocker. These data suggest that the blockade of voltage-gated sodium channels is a possible mechanism of action for the well-known neuroprotective and anticonvulsant properties of vinpocetine.

Characterization of a 5.4 kb cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases.

Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70-80 kb. Although titin has been studied by several laboratories, barely more than half of the cDNA sequence (approximately 45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule). A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in order to obtain sequence data from titin's unknown N-terminal region. A monoclonal antibody (T12), which binds to an epitope close to the Z-line, was used to identify initial cDNA clones. Additional overlapping clones were isolated and sequenced yielding a 5.4 kb contig. The encoded polypeptide contains 16 Type-II domains and four unique intervening segments. Polyclonal sera, raised against an expressed protein fragment encoded by the 5' end of the contig, strongly stained the Z-line of myofibrils of different species. However, the sequence of this fragment is 83% identical at the amino acid level with the previously reported C-terminal (i.e. M-line) end of chicken embryonic skeletal muscle titin. The expressed protein fragment could be phosphorylated in vitro by embryonic skeletal muscle extract and by the purified proline-directed kinase ERK1, presumably at the xSPxR recognition sites located in the first interdomain segment.

The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.

The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin.

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA.

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

Nuclear import of DNA in digitonin-permeabilized cells.

DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.