Neurodegenerative Changes in the Structural and Ultrastructural Organization in the Pyriform Cortex of 5xFAD Transgenic Mice

Journal of Evolutionary Biochemistry and Physiology - Analysis of pathological changes in the structural and functional organization of the pyriform cortex in mice of the 5xFAD line, which models...     

Development of lucerne (Medicago sativa L.) treated with mineral fertilizer and manure at optimal and water deficit conditions

A study on the effect of different rates of mineral fertilizer and manure on yield parameters of lucerne under optimal and water deficit conditions was carried out. Leached chernozem soil and lucerne cultivar Victoria were used. The soil was treated with ammonium nitrate and fully matured cattle manure. The plants were grown under optimum moisture content of 80% and 40% of field capacity. The water deficit stress decreased top and root biomass by 11-75% and 3-29% at mineral and organic fertilization, respectively. The applied mineral and organic N strongly depressed nodules development. Both mineral fertilizer and organic manure at dose of 210 mg N kg(-1) soil completely inhibited the appearance of nodules. Next to nitrogen, water deficit stress further inhibited the development of nodules. Nitrogen fertilization increased seed productivity in the two experimental moisture conditions. The water deficit stress decreased seed productivity by 18 to 33% as compared to optimum conditions. The plant treatments with manure were much more resistant to water deficit and recovering ability of plants was faster as compared to treatments with mineral fertilizer. The application of manure stimulates development of drought-stress tolerance in lucerne. However, the results obtained can be considered for the soil type and experimental conditions used.     

Optimization of BY‐2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method

We have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY‐2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO₃, NH₄NO₃ and CaCl₂ and the hormones 2,4‐dichlorophenoxyacetic acid (2,4–D) and 6‐benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO₃, NH₄NO₃ and CaCl₂) and also individually between 2,4‐D and three other components (KNO₃, NH₄NO₃ and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 μg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20‐fold (day 7) and 31‐fold (day 10) compared to the conventional MS medium.     

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c ₂, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c ₂ molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His₁₂-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c ₂ is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His₁₂-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c ₂ to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.     

Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis

Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.     

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.     

Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen

The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.     

Ultrastructural localization of the membrane-bound Mg-adenosine triphosphatase activity in rat meninges

The distribution of the membrane-bound magnesium ions-dependent adenosine triphosphatase (Mg-ATPase) activity has been studied ultracytochemically in rat meninges by the method of Wachstein and Meisel (1957). A device specially constructed to avoid preparation artefacts has been used to obtain sections from the parietal region of the head. The meninges display an intense though irregularly distributed ATPase activity marked by depositions of electron-dense reaction product (RP) which is almost absent in the outer and middle dural layers. In the borderline zone between dura mater and the arachnoid the RP deposits are found at the outer surface of the inner dural cells and at the contact sites between these cells and the dural neurothelium. The intercellular cleft(s) between the neurothelium and the outer arachnoidal layer, occupied by an "electron-dense band", remains free of RP. The strongest accumulations of reactions granules are observed on the surface of the leptomeningeal cells of the arachnoidal space. In the contact region between the inner arachnoidal and the outer pial layers the distribution of the RP is similar to the one observed in the interface zone dura mater/arachnoid, while the pial cells themselves are definitely reaction-positive. In all meningeal vessels RP is found at the lumenal and abluminal aspects of the endothelium as well as at the cell membranes of the perivascular cells. These results emphasize the importance of the dural neurothelium for the functions of the blood-cerebrospinal fluid (CSF)-barrier between the dural blood vessels and the CSF.     

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.