Conformation of Bovine Nerve Root P2 Protein: Characteristics of the Molecule from Circular Dichroism Spectra

Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P₂ basic protein, its reduced and carboxymethylated derivative (RCM-P₂), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM-P₂ have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino- and carboxy-terminal segments of the P₂ protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P₂ protein in aqueous solutions. The P₂ protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P₂ protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P₂ protein is denatured with 5 M urea.     

Flanking Regulatory Sequences of the Tetrahymena R Deletion Element Determine the Boundaries of DNA Rearrangement

In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.     

An in vitro assessment of the effects of broad-spectrum antibiotics on the human gut microflora and concomitant isolation of a Lactobacillus plantarum with anti-Candida activities

Chemostat culture was used to determine the effects of the antimicrobial agents tetracycline and nystatin on predominant components of the human gut microflora. Their addition to mixed culture systems caused a non-specific, and variable, decrease in microbial populations, although tetracycline allowed an increase in numbers of yeasts. Both had a profound inhibitory effect upon populations seen as important for gut health (bifidobacteria, lactobacilli). However, a tetracycline resistant Lactobacillus was enriched from the experiments. A combination of genotypic and phenotypic characterisation confirmed its identity as Lactobacillus plantarum. This strain exerted powerful inhibitory effects against Candida albicans. Because of its ability to resist the effects of tetracycline, this organism may be useful as a probiotic for the improved management of yeast related conditions such as thrush and irritable bowel syndrome.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.