A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.     

Characterization of growth hormone binding sites in granulosa and theca layers at different stages of follicular maturation and ovulatory cycle in the domestic hen

The currently available evidence points to a possible influence of growth hormone (GH) on avian folliculogenesis, which can be mediated by both hepatic- and ovarian-derived IGF-I. Therefore, the purpose of the present study was to reveal GH-binding sites in granulosa and theca layers of preovulatory follicles and to determine the binding characteristics depending on the degree of follicular maturation and the stage of the ovulatory cycle in the hen. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition, and the five largest yellow follicles (from F1 to F5) were removed. GH-binding sites in granulosa and theca layers from F1 to F5 follicles were characterized using a radioreceptor assay. Equilibrium dissociation constants (K(d)) and binding capacities (B(max)) were determined by Scatchard analysis of saturation curves, which revealed a single class of high-affinity GH-binding sites in both theca tissue and granulosa cells. In F1, F2, and F5 follicles, B(max) and K(d) for GH-binding sites in the granulosa layer changed during the ovulatory cycle, decreasing between stages I and III, to increase again at stage IV, with alterations in K(d) being less profound. No significant differences in binding capacities and affinities of GH-binding sites in the theca layer were found between various stages of the cycle. Furthermore, the concentration of GH-binding sites in the granulosa layer rose, whereas that in the theca layer fell with follicular enlargement. These findings indicate the presence of high-affinity GH-binding sites in both granulosa and theca layers of hen preovulatory follicles. Data also demonstrate that GH-binding sites in these tissues are regulated in a tissue-specific manner. Furthermore, the regulation of binding capacity of GH binding in granulosa cells by hormonal factors associated with ovulatory cycle is apparently not dependent on the state of follicular maturation.     

Antibody specific for the peptide.major histocompatibility complex. Is it T cell receptor-like?

Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.     

Carbonic Anhydrase of the Alkaliphilic Cyanobacterium Microcoleus chthonoplastes

The activity of carbonic anhydrase (CA) was studied in different cell fractions of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The activity of this enzyme was found in the soluble and membrane protein fractions, as well as in intact cells and in a thick glycocalyx layer enclosing the cyanobacterium cells. The localization of CA in glycocalyx of M. chthonoplastes was shown by western blot analysis and by immunoelectron microscopy studies with antibodies to the thylakoid CA from Chlamydomonas reinhardtii (Cah3). At least one of the CA forms occurring in M. chthonoplastes CA was shown to be an -type enzyme. A possible mechanism of the involvement of the glycocalyx CA in calcification of cyanobacteria is discussed.     

Influence of different F1-specific components of Yersinia pestis capsular antigen on cell-mediated immunity

The F1-specific components of Y. pestis capsular antigen, isolated by Baker's method, were shown to differ by their biological activity and the character of action on cell-mediated immunity factors, used in this study. Depending on the method of isolation, antigens could vary in the proportion of their components, which determined the specific features of the total preparations obtained in this investigation. Out of the four components under study having the same antigenic specificity, but different physico-chemical characteristics and genetically determined synthesis, only one component exhibited biological properties dominating in the secreted form of F1, more similar in their composition to protein Caf1. The other three components exhibited immunological activity on the level of unspecific protection in different ways, depending on the model, and influencing the outcome of the interaction "bacteria-macrophage". Lectin-like hemagglutinating and hemolyzing activity was shown by components not related to protein Caf1. The multi-component character of preparations F1 (Baker) and the individual activity of their components should be taken into consideration when using capsular antigen and different methods of its isolation for different aims.     

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.     

Characteristics of the functional state of rat tracheal and bronchial smooth muscle at different stages of alveolitis

In isolated tracheal smooth muscle preparations in normal rats and in rats with experimental fibrotic alveolitis, responses to electrical field stimulation of nervous and muscle fibers were studied. At stimulation of muscles or nerves of tracheal preparations without intramural ganglia in rats with acute alveolitis, parameters of smooth muscle contractions did not practically differ from those in normal rats. In rats with fibrotic alveolitis the amplitude and rate of muscle contraction decreased, while the response latent period (LP) increased. At stimulation of preganglionic nerve fibers of the tracheal preparations with intramural ganglia in rats with acute alveolitis, the value and rate of smooth muscle contraction decreased, while the response LP increased. After transition into chronic phase of the disease (fibrotic alveolitis), a partial restoration of the response parameters took place. In rats with acute alveolitis, the repeated stimulation of the nerve fibers led to an increase of amplitude and a decrease of rate of tracheal smooth muscle contractions. In rats with fibrotic alveolitis, the repeated stimulation caused a decrease of amplitude and rate of contractions and an increase of the response LP.     

Methods of diagnosis and differentiation of plague pathogen: approaches to detection of atypical strains of Yersinia pestis by molecular biology. Part I

A review of data on the modern methods of detection of typical and atypical strains of the plague agent Y. pestis is given. The history of the development of the molecular-biological tests for the differentiation of Y. pestis from the related microorganisms is presented. The problems facing investigators during the development of these tests are discussed.     

Genetic control of the wheat Triticum monococcum L. resistance to powdery mildew

Using hybrid analysis and test-clone method, 102 accessions of Triticum monococcum L. from the collection of the Vavilov All-Russia Institute of Plant Industry have been studied. This species of wheat has been found to by considerably polymorphic with respect to the resistance to the fungus Erysiphe graminis DC. f. sp. tritici Marchal. causing powdery mildew. The resistance of most accessions to the fungus population and clones is determined by dominant genes. In rare cases, the resistance was determined by recessive genes or one, two, or three oligogenes. A group of einkorn wheat accessions has been found in which the resistance to powdery mildew was determined by the same dominant factor or different but closely linked ones. Recessive resistance genes of T. monococcum differ from the recessive gene pm5 determining the resistance of T. aestivum plants. The genome of T. monococcum contains various genes of resistance to powdery mildew and is a potential source of effective genes to be used when selecting cultivated species of wheat for immunity.