Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.     

Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3'-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.     

Subjective effects of peak clipping and compression limiting in normal and hearing-impaired children and adults

Despite many advances in hearing-aid signal processing, compression limiting and peak clipping are still used. To date, perceptual studies have been conducted only with adults. The current study was designed to investigate the clarity of peak-clipped and compressed speech for both adults and children. Subjects were 30 normal-hearing and 30 hearing-impaired individuals in three age ranges (7-9, 10-12, and 16-50 years). Stimuli were processed at 60, 70, 75, and 80 dB SPL using peak clipping and at 80 dB SPL using compression limiting. Paired-comparison measures were used to assess the clarity of sentences, and a signal-to-distortion ratio (SDR) based on a measure of coherence between input and output was computed for each condition. For the peak-clipping conditions, there was a decrease in perceived clarity as the input increased from 60 to 80 dB SPL. This perceptual continuum was most apparent for the normal-hearing adults. The normal-hearing 10-12 year olds and the hearing-impaired adults showed a similar, but less pronounced, pattern. In contrast, the remaining three subject groups showed minimal differences in perceived clarity across conditions. Surprisingly, only the two oldest normal-hearing groups showed a clear preference for compression limiting over peak clipping at the highest input level, and only their results were consistent with the pattern of coherence across stimuli. Judgments of clarity by the normal-hearing subjects correlated best with the SDR in the 500-2000-Hz range, while clarity judgments of the hearing-impaired subjects correlated best with the SDR below 1000 Hz.     

Allosteric gating of a large conductance Ca-activated K+ channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

Interaction between proteins containing homeodomains associated to leucine zippers from sunflower

A strategy based on the use of PCR with one degenerate oligonucleotide deduced from conserved sequences and lambda gt10 primers was used to isolate homeobox containing sequences from sunflower stem and root cDNA libraries. Six different partial cDNAs coding for the first 48 amino acids of homeodomains and amino terminal sequences were analyzed and found to be members of the HD-Zip superfamily, which contain a homeobox linked to a leucine zipper coding region. A full-length cDNA clone, Hahb-10, was isolated and characterized. The leucine zipper portions of Hahb-10 and of the previously reported Hahb-1 have been utilized to construct fusions with the N-terminal domain of the lambda repressor. These fusions were tested for their ability to bind to lambda promoters in vivo. The expression of a protein containing an active dimerization domain, but not capable of DNA binding, exerts a dominant negative effect on the ability of repressor-zipper fusions to bind to its target DNA. From these experiments, it was concluded that Hahb-1 and -10, when co-expressed, form preferentially homodimers. Exchange of conserved threonines and leucines at positions a1 and d1 of both zippers reduces dimerization efficiency and allows the formation of heterodimers, suggesting that these residues are, among others, determinants of the specificity of interaction, most likely through changes in hydrophobic packing interactions at the dimer interface. The results imply that a great number of interacting molecular entities compose this protein superfamily which is presumably involved in regulating plant developmental responses.