Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

The behavioral and biochemical sequelae of the removal of the olfactory bulbs in C57Bl/6j mice]

Ablation of the olfactory bulbs in mice C57Bl/6j was accompanied by motor and orienting-exploratory activity augmentation in the "open field" test and deterioration of the learning ability to active and passive avoidance. The most expressed behavioural changes developed in four weeks after the surgery. Chronic administration of antidepressants (amitriptyline, 20 mg/kg; trazodone, 20 mg/kg; imipramine, 10 mg/kg; intraperitoneally) normalized behaviour of bulbectomized animals, trazodone being the most effective. In the biochemical studies the brainstem serotonin level was found to be decreased and the density of 5HT2-receptors in the cerebral cortex increased in bulbectomized mice. Only trazodone was able to correct the biochemical indices. The state of the bulbectomized mice is supposed to serve a model of a depression with hypo-function of serotonergic system of the brain.     

Comparative evaluation of methacycline hydrochloride capsules and tablets according to their dissolubility and rate of solution]

Desintegration and dissolution of capsules and tablets of methacycline hydrochloride were studied. The study on solubility of methacycline hydrochliride capsules filled with methacycline granulate or powder according to the same formula showed that the rate of the antibiotic liberation from the capsules filled with the powder decreased during storage while that from the capsules filled with the granulate did not change. Investigation of the effect of the mass packing value in a drop on the antibiotic liberation from the capsules showed that an increase in the packing coefficient above 1.38 resulted in a marked decrease in the rate of methacycline liberation from the capsules filled with the granulate. No correlation between desintegration and dissolution of methacycline capsules and tablets was found.     

The autophosphorylation reaction in the mechanism of activation of pig brain cyclic AMP-dependent protein kinase

Autophosphorylation of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phospho form of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. The activation constant of phospho- and dephosphoprotein kinase under the influence of cyclic AMP and the dissociation constant of the cyclic AMP complex with phospho- and dephospho forms of the holoenzyme were estimated. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and non-phosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cycli AMP. The anti-conformation of the cyclic nucleotide fixed in the complex with the phospho form of the regulatory subunit is suggested.     

Antibiotic sensitivity of the H- and O-forms of bacteria in the genus Proteus]

Proteus strains isolated from the gastro-intestinal tract of children not older than 1 year were characterized by resistance to oxytetracycline, chlortetracycline, benzylpenicillin and erythromycin. The strains were more sensitive to neomycin, monomycin and streptomycin. Antibiotic sensitivity of Pr. mirabilis and Pr. vulgaris strains increased on transfer from H- to O-form. Inverse dependence of the urease activity of the strains on their sensitivity to tetracyclines was noted.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Nanoscale iron(III) oxyhydroxy aggregates formed in the presence of functional water-soluble polymers: models for iron(III) biomineralisation processes

Superparamagnetic clusters of iron(III) oxyhydroxide in the form of poorly crystalline ferrihydrite (formally, 5Fe2O3 x 9H2O) have been synthesised in the presence of the polymers polyvinyl alcohol (PVA), polyacrylic acid (PAA) and alginic acid. The solutions have been characterised by viscosity studies and the resultant arrays isolated from these solutions have been imaged under an electron microscope and their magnetic properties determined by 57Fe-M?ssbauer spectroscopic studies at 293 and 77 K and magnetisation measurements at 293 and 5 K. The magnetic data show that the iron(III) oxyhydroxy particles are superparamagnetic. All preparations show hysteretic behaviour with coercive fields being approximately half or less than half of that of ferrihydrite (3.4 kOe) and values of magnetic moment per iron particle less than that of ferrihydrite. Nanoscale aggregates (2-4 nm) are formed in the presence of PVA and PAA while, with alginic acid, extended branch-like structures are observed, their formation being facilitated by the comparatively rigid polysaccharide chain, a process related to iron biomineralisation in diverse biological systems.     

Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RIalpha subunit

The primary mediator of cAMP action in mammalian cells is cAMP-dependent protein kinase (PKA). There are two types of PKA, type I (PKA-I) and type II (PKA-II), which share a common catalytic subunit but contain distinct regulatory subunits, RI and RII, respectively. Evidence suggests that increased expression of RIalpha/PKA-I correlates with neoplastic cell growth. Here, we show that sequence-specific oligonucleotide inhibition of RIalpha expression results in inhibition of growth and modulation of cAMP signaling in cancer cells. The antisense promoted growth inhibition in a time-dependent, concentration-dependent, and sequence-dependent manner in human cancer cells in monolayer culture, and it inhibited colony formation in soft agar and tumor growth in nude mice. Among the cancer cells are LS-174T, HCT-15, and Colo-205 colon carcinoma cells; A-549 lung carcinoma cells; LNCaP prostate adenocarcinoma cells; Molt-4 leukemia cells; and Jurkat T lymphoma cells. Northern blot and immunoprecipitation analyses revealed that the growth inhibitory effect of the antisense correlated with a decrease in RIalpha expression at both the mRNA and protein levels. Pulse-chase experiments revealed that the antisense-directed inhibition of RIalpha expression resulted in compensatory changes in expression of the isoforms of R and C subunits and cAMP signaling in a cell type-specific manner. These results demonstrate that cAMP is ubiquitous in the regulation of cell growth and that the antisense oligonucleotide, which inhibits the synthesis of the RIalpha subunit of PKA, can be targeted to a single gene for treatment of cancer in a variety of cell types.