Treatment of diabetes in NOD mice by gene transfer of Ig-fusion proteins into B cells: role of T regulatory cells

We previously reported that retrovirally mediated gene expression of Ig fusion proteins leads to specific immunologic tolerance and successful treatment of autoimmune conditions. Thus, a single dose of GAD65-IgG- or (Pro) Insulin-IgG-transduced B cells delays the onset and decreases the incidence of diabetes in young (7-12 weeks old) NOD female mice. Herein, we tested the role of regulatory T cells by in vivo treatment with anti-CD25 before B-cell gene therapy or by in vitro ablation of CD25+ cells from tolerized hosts in an adoptive transfer model. Our results demonstrate that anti-CD25 treatment, like cyclophosphamide, partially blocks the efficacy of gene therapy for tolerance. Moreover, B-cell therapy is effective at preventing diabetes transfer by female T cells (from older diabetic mice) into intact male recipients with normal islets, but failed to do so in NOD-scid recipients. This is due in part to homeostatic proliferation but also to the absence of CD25+ T cells in the latter hosts. Tolerance induced in younger NOD females can be stably transferred to NOD-scid recipients. However, physical removal of CD25+ cells abrogates the transfer of tolerance. Therefore, we conclude that CD4+, CD25+ regulatory T cells are required for the induction as well as maintenance of tolerance in this gene therapy model. The phenotype of these induced regulatory T cells is under investigation.     

Synthesis of triterpenoid acylates - an effective reproduction inhibitors of influenza A (H1N1) and papilloma viruses

The synthesis of a new group of triterpenoid acylates on the basis of oleanolic, glycyrrhetic and ursolic acids and betulin is described. In studying the activity of the synthesized compounds in relation to reproduction of virus pathogens of respiratory infections 28-O-methoxycynnamoylbetulin shows high activity against influenza type A (H1N1) the selectivity index SI > 100. The high activity of 3,28-dinicotinoylbetulin against papilloma virus (strain HPV-11) was detected, the selectivity index SI was 35.     

Neuronal activity in a honey bee (Apis mellifera L.) with kynurenine deficiency

Neuronal activity of the antennal lobes, mushroom bodies, and cervical connective in wild-type honey bees and snowlaranija mutants was recorded at different stages of the ontogeny (on the 1st, 3rd, 7th, and 25th days). The mutation snowlaranija affects the structural gene of tryptophane oxygenase, the first key exzyme in the kynurenine pathway of tryptophane metabolism, and leads to a deficit of kynurenines. Changes in neuronal activity in nutant bees were most pronounced in the cervical connective. A significant decrease in the pulse rate was revealed only in homozygous but not in heterozygous individuals. This finding is in accordance with previously reported inhibitory effect of the mutation at the behavioral level. Less pronounced effects were obtained when the neuronal activity was recorded in the antennal lobes or mushroom bodies. This may be related to a complex character of biochemical changes in different parts of mutants brain.     

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.     

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.     

Fractionation and specificity of DNA methylases from the Escherichia coli SK cells

Summary Specificity of DNA methylation enzymes from the E. coli SK cells and conditions for their separation have been investigated. Column chromatography on carboxymethylcellulose permits fractionation of methylase activity into six discrete peaks whose specificity to the methylated base has been determined in vitro with H³-SAM as precursor. All methylases specific for adenine produced 6-methylaminopurine, all methylases specific for cytosine yielded 5-methylcytosine.The first enzymatic activity peak containing cytosine methylase free of traces of adenine-methyiating activity (E₁), and the second peak containing both the enzymes (E₂) were not adsorbed on the ion exchanger and went off the column with the effluent (column buffer). Adenine specific methylase E₂ is retarded to a small extent during the passage through the column. The second adenine methylases (W) was characterized by weak bonds with the ion exchanger and was removed when washing the column with column buffer. The elution with NaCl gradient produced successively three enzymatic activity peaks: adenine methylase (G_I), cytosine methylase (G_II), and one more adenine methylase (G_III) removed from the column by 0.16 m, 0.24 m and 0.43 m NaCl respectively.Using a new modification of the complementary methylation test, the specificity with regard to recognition site was examined for all enzymes, except for W and G_III, which were extremely unstable. The adenine methylases E₂ and G_I and the cytosine methylases E₁ and G_II were shown to recognize different sites and to be different enzymes. In view of the drastic differences in their chromatographic behaviour and physical stability, the adenine methylases W and G_III are probably also different enzymes.     

3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge

MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.     

Effect of prenatal emotional-pain stress on the status of interphase chromatin in neurons of the rat developing brain with various excitation of the nervous system

A short-term emotional-painful stress, experienced by pregnant rat females differing in threshold of excitability of their nervous system, was used to assess the state of interphase condensed chromatin and C-heterochromatin of neuron nuclei in developing brains of 16-17 day old embryos. To reveal relationships between the genetically determined excitability of rats and the state of interphase chromatin in their neuron nuclei a computer information system has been used that enabled us to classify the neuronal nuclei according to their specific DNA image cytometry features. The results indicate an obvious relationship between excitability of the nervous system and structural-functional state of the neuronal interphase chromatin.