Formation of Liquid-Crystalline Dispersions of Double-Stranded DNA–Chitosan Complexes

Right-handed helical double-stranded DNA molecules were shown to interact with chitosans to form under certain conditions (chitosan molecular weight, content of amino groups, distance between amino groups, ionic strength and pH of solution) cholesteric liquid-crystalline dispersions characterized by abnormal positive band in CD spectrum in the absorption region of DNA nitrogen bases. Conditions were found for the appearance of intense negative band in CD spectrum upon dispersion formation. In some cases, no intense band appeared in CD spectrum in spite of dispersion formation. These results indicate not only the multiple forms of liquid-crystalline dispersions of DNA–chitosan complexes but also a possibility to control the spatial properties of these complexes. The multiplicity of liquid-crystalline forms of DNA–chitosan complexes was attempted to explain by the effect of character of dipoles distribution over the surface of DNA molecules on the sense of spatial twist of cholesteric liquid crystals resulting from molecules of the complexes.     

Calcium transients in the model of rapidly induced anoxic tolerance in rat cortical slices: involvement of NMDA receptors

In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca(2+) concentration (Ca(i)) and in the level of Ca(2+) bound to intracellular hydrophobic domains (Ca(b)) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Ca(i) and Ca(b), reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca(2+) pools and prevents Ca(2+) overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.     

Trehalose catabolism in microsporidia Nosema grylli spores

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Hydration and structure of hemiprotonated polycytidylic acid in films

Structural transitions of poly(rC)-Ka+ in humid films with different water content were studied by infrared spectroscopy and piezogravimetry. From analysis of the hydration isotherms and the dependence of spectral parameters (frequencies and intensities of the main bands) on n the hydration sites of the polynucleotide were determined (C2O, O4', N4H2, N1, PO2-, C2'OH). It was found that the transition of the polynucleotide from the unordered state to a double-stranded complex poly(rC+).poly(rC) occurs in the interval of n from 2 to 8. The value n = 8 corresponds to the total hydration of poly(rC). A model of hydration of poly(rC+).poly(rC) based on the experimental results and known X-ray parameters of this double helix complex is proposed. The most important feature of the model is the presence of single water bridges between PO2(-)-groups in the first hydration shell of each chain and triple water bridges between O4', N4H2 and C2'OH- atomic groups of opposite chains. The experimental results obtained and the proposed structure of hydration environment of poly(rC+).poly(rC) suggest that the stabilization of this complex is stabilized by the intra- and inter-chain water bridges and hydrogen bonds between pairs of cytosine bases.     

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.     

Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.)

The mitochondrial DNA (mtDNA) of individuals from 79 colonies of Apis mellifera from five Canary Islands was studied using the Dra I test based on the restriction of PCR products of the tRNAleu–COII intergenic region. Five haplotypes of the African (A) lineage and one of the west European (C) lineage were found. The haplotypes A14 and A15 are described for the first time. These haplotypes have a new P sequence named P₁. The wide distribution and high frequency of haplotype A15 suggest that it is characteristic of the Canarian Archipelago. Sources of haplotype variability of honeybee mtDNA in the Canary Islands (waves of colonization from Africa, queen importations, habitat diversification) are discussed.     

Chromosomal interrelations in the interphase nucleus

The distribution of prekinetochores in human lymphocytes has been studied by indirect immunofluorescence with autoantibodies against kinetochore. Lymphocyte flattening that allowed a 5-6 increase in their size, was suggested in addition to a method of lymphocyte stretching allowing a 10-fold extension. Prekinetochores in flat and stretched lymphocytes are seen settled down as separate pairs. The equal pattern of staining of these prekinetochores in each pair suggests that homologous chromosomes located in pairs.