Cyclodextrin-modified polyethylenimine polymers for gene delivery

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.     

New hepatitis B virus of cranes that has an unexpected broad host range

All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.     

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.     

Human recombinant anti-La (SS-B) autoantibodies demonstrate the accumulation of phosphoserine-366-containing la isoforms in nucleoplasmic speckles

Using the recombinant La (SS-B) protein or a phosphorylated peptide derived thereof 27 La-specific human recombinant autoantibodies were selected from anti-La-positive systemic lupus erythematosus and systemic sclerosis patient-derived combinatorial phage display antibody libraries. Binding of these anti-La antibodies to various isoforms of the La protein present in normal and apoptotic cell extracts was analysed by Western blotting. Twenty-four of the selected antibodies recognize most, if not all isoforms of La, whereas three are exclusively reactive with the protein phosphorylated at serine-366. Sequence analysis of the selected antibodies showed a restricted spectrum of diversity in their VH germline gene usage. Remarkably, the recombinant antibodies recognizing exclusively the phosphoserine-366-containing isoform of La displayed a spleckled nucleoplasmic staining pattern in immunofluorescence analysis of HeLa and HEp-2 cells. This pattern differed markedly from those obtained with anti-La antibodies recognizing all isoforms of the La protein. Colocalization experiments with marker antibodies for spliceosomal UsnRNPs and RNA polymerase III subunits revealed that the anti-phosphorylated La antibodies stain the same nucleoplasmic speckles as anti-UsnRNP antibodies. In contrast to anti-UsnRNP antibodies the anti-phosphorylated La antibodies did not stain the Cajal bodies. In addition, no colocalization of phosphorylated La with RNA polymerase III was observed. Potential functional implications of the accumulation of phosphorylated La in nucleoplasmic speckles are discussed.     

Gene expression profiling on global cDNA arrays gives hints concerning potential signal transduction pathways involved in cardiac fibrosis of renal failure

Cardiac remodelling with interstitial fibrosis in renal failure, which so far is only poorly understood on the molecular level, was investigated in the rat model by a global gene expression profiling analysis. Sprague-Dawley rats were subjected to subtotal nephrectomy (SNX) or sham operation (sham) and followed for 2 and 12 weeks, respectively. Heart-specific gene expression profiling, with RZPD Rat Unigene-1 cDNA arrays containing about 27 000 gene and EST sequences revealed substantial changes in gene expression in SNX compared to sham animals. Motor protein genes, growth and differentiation markers, and extracellular matrix genes were upregulated in SNX rats. Obviously, not only genes involved in cardiomyocyte hypertrophy, but also genes involved in the expansion of non-vascular interstitial tissue are activated very early in animals with renal failure. Together with earlier findings in the SNX model, the present data suggest the hypothesis that the local renin-angiotensin system (RAS) may be activated by at least two pathways: (a) via second messengers and Gproteins (short-term signalling); and (b) via motor proteins, actins and integrins (longterm signalling). The study documents that complex hybridization analysis yields reproducible and promising results of patterns of gene activation pointing to signalling pathways involved in cardiac remodelling in renal failure. The complete array data are available via http://www.rzpd.de/cgi-bin/services/exp/viewExpressionData.pl.cgi.     

The effect of attempted ballistic training on the force and speed of movements

Athletes in sports requiring explosive movements might benefit from a unique form of training in which a limb is restrained while the athlete attempts ballistic (explosive) movements. We investigated the effects of such ballistic training and conventional resistance training on force and speed of front kicks, side kicks, and palm strikes of martial artists. We assigned subjects randomly to an experimental group (n = 13) or a control (normal martial art training) group (n = 9). Conventional resistance training produced a gain of 12% (95% likely limits +/- 13%) in front kick force relative to the control group. Overall ballistic training and conventional resistance training decreased side kick force by 15% (+/-14%), but movement speeds increased by 11-21% (+/-13-17%). Responses to ballistic training were generally more marked in more highly skilled athletes. Attempted ballistic training may be a beneficial adjunct to resistance training for skilled athletes in sports where speed rather than force is critical.