Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system.

An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects

Background

Although influenza causes significant morbidity and mortality in the elderly, the factors underlying the reduced vaccine immunogenicity and efficacy in this age group are not completely understood. Age and immunosenescence factors, and their impact on humoral immunity after influenza vaccination, are of growing interest for the development of better vaccines for the elderly.

Methods

We assessed associations between age and immunosenescence markers (T cell receptor rearrangement excision circles – TREC content, peripheral white blood cell telomerase – TERT expression and CD28 expression on T cells) and influenza A/H1N1 vaccine-induced measures of humoral immunity in 106 older subjects at baseline and three timepoints post-vaccination.

Results

TERT activity (TERT mRNA expression) was significantly positively correlated with the observed increase in the influenza-specific memory B cell ELISPOT response at Day 28 compared to baseline (p-value=0.025). TREC levels were positively correlated with the baseline and early (Day 3) influenza A/H1N1-specific memory B cell ELISPOT response (p-value=0.042 and p-value=0.035, respectively). The expression and/or expression change of CD28 on CD4+ and/or CD8+ T cells at baseline and Day 3 was positively correlated with the influenza A/H1N1-specific memory B cell ELISPOT response at baseline, Day 28 and Day 75 post-vaccination. In a multivariable analysis, the peak antibody response (HAI and/or VNA at Day 28) was negatively associated with age, the percentage of CD8+CD28low T cells, IgD+CD27- naïve B cells, and percentage overall CD20- B cells and plasmablasts, measured at Day 3 post-vaccination. The early change in influenza-specific memory B cell ELISPOT response was positively correlated with the observed increase in influenza A/H1N1-specific HAI antibodies at Day 28 and Day 75 relative to baseline (p-value=0.007 and p-value=0.005, respectively).

Conclusion

Our data suggest that influenza-specific humoral immunity is significantly influenced by age, and that specific markers of immunosenescence (e.g., the baseline/early expression of CD28 on CD4+ and/or CD8+ T cells and T cell immune abnormalities) are correlated with different humoral immune response outcomes observed after vaccination in older individuals, and thus can be potentially used to predict vaccine immunogenicity.     

A model for switch-like phenomena in biological systems.

We present a model for the activity of protein clusters based on a simultaneous desorption of an activator (agonist, substrate molecule, etc.) and an inactivator (antagonist, inhibitor, etc.) caused by the collision or interaction between two effector molecules (e.g. receptors, enzymes). This model gives rise to switch-like dose-response curves, which are difficult to explain by ordinary co-operativity. It fits with recent experimental results obtained on single cells. Some other interesting aspects of the model are also pointed out. The model is similar to the model used to explain steep 'dose-response curves' in heterogeneous catalysis, caused by the reaction between two different molecules or atoms on the surface of the catalyst.     

Induction and characterization of Pediococcus acidilactici temperate bacteriophage

Mitomycin C was used to induce temperate bacteriophage from three strains of Pediococcus acidilactici. The new bacteriophage, designated pa97, pa40, and pa42, were characterized based on morphology, DNA homology, and major protein profiles. Morphological attributes (small isometric heads with non-contractile tails) place these bacteriophages within the B1 group of the family Siphovirdae. Restriction endonuclease digests suggested that the bacteriophage genomes were linear molecules without cohesive ends, and between 33 and 37 kilobases in length. All three bacteriophages possessed one major protein with an estimated mass of 30 to 35 kilodaltons. Bacteriophage pa42 also contained a second major protein of approximately 47 kilodaltons. DNA-DNA hybridization showed bacteriophages pa40 and pa42 were homologous to each other, but not to pa97, suggesting that Pediococcus acidilactici bacteriophage fall into at least two different species.     

Dexamethasone acts as a negative regulator of epidermal growth factor receptor synthesis in fetal rat lung cells

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung cells is decreased by approximately 50% following 24-h dexamethasone treatment. Ligand binding assays identified an average of 30,000 receptors per cell in untreated FRL cells, while analysis of dexamethasone treated cells showed a decrease to about 16,000 receptors per cell. No substantial changes in receptor affinities were detected. Immunoprecipitation of 35S-methionine-labeled EGF receptor protein demonstrated a 50% decrease in total EGF receptor protein after 24-h dexamethasone treatment. Brief pulse labeling with 35S-methionine showed that the reduction in total EGF receptor protein content was due to a decrease in EGF receptor synthesis. Receptor synthesis declined about 25% after 1 h of dexamethasone treatment and at 3 h, EGF receptor synthesis was maximally decreased to nearly 50% that of cells not exposed to dexamethasone. Dexamethasone treatment was also effective in reducing EGF receptor synthesis in cells pretreated with retinoic acid, an agent which enhances receptor synthesis. These data are the first to document a dexamethasone-induced decrease in EGF receptor synthesis. Furthermore, these findings may provide a plausible mechanism by which dexamethasone could regulate EGF responsiveness.