Reconstitution of the Na+K+ pump of Ehrlich ascites tumor and enhancement of efficiency by quercetin

Plasma membranes from Ehrlich ascites tumor cells were solubilized by octylglucoside in the presence of phospholipids. The Na+K+-ATPase was purified from this extract by adsorption and elution from thio-Seph-arose 4B. The enzyme (specific activity, 7 mumoles of ATP hydrolyzed min-1 mg of protein -1) was reconstituted into liposomes by the octyglucoside dilution procedure. An ATP-dependent Na+ influx with low efficiency was observed. On addition of appropriate amounts of quercetin, the Na+ flux/ATP hydrolysis ratio was increased from 0.4 to 1.4.     

A test of two mechanisms proposed to optimize grassland aboveground primary productivity in response to grazing

Aims Mesic grasslands have a long evolutionary history of grazing by large herbivores and as a consequence, grassland species have numerous adaptations allowing them to respond favourably to grazing. Although empirical evidence has been equivocal, theory predicts that such adaptations combined with alterations in resources can lead to grazing-induced overcompensation in aboveground net primary production (ANPP; grazed ANPP> ungrazed ANPP) under certain conditions. We tested two specific predictions from theory. First, overcompensation is more likely to occur in annually burned grasslands because limiting nutrients that would be lost with frequent fires are recycled through grazers and stimulate ANPP. Second, overcompensation of biomass lost to grazers is more likely to occur in unburned sites where grazing has the greatest effect on increasing light availability through alterations in canopy structure.Methods We tested these nutrient versus light-based predictions in grazed grasslands that had been annually burned or protected from fire for>20 years. We assessed responses in ANPP to grazing by large ungulates using both permanent and moveable grazing exclosures (252 exclosures from which biomass was harvested from 3192 quadrats) in a 2-year study. Study sites were located at the Konza Prairie Biological Station (KPBS) in North America and at Kruger National Park (KNP) in South Africa. At KPBS, sites were grazed by North American bison whereas in KNP sites were grazed either by a diverse suite of herbivores (e.g. blue wildebeest, Burchell's zebra, African buffalo) or by a single large ungulate (African buffalo).Important findings We found no evidence for overcompensation in either burned or unburned sites, regardless of grazer type. Thus, there was no support for either mechanism leading to overcompensation. Instead, complete compensation of total biomass lost to grazers was the most common response characterizing grazing–ANPP relationships with, in some cases, undercompensation of grass ANPP being offset by increased ANPP of forbs likely due to competitive release. The capability of these very different grass-dominated systems to maintain ANPP while being grazed has important implications for energy flow, ecosystem function and the trophic dynamics of grasslands.     

Azithromycin Treatment Alters Gene Expression in Inflammatory,Lipid Metabolism,and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides.     

Cell division in morphological mutants ofAgmenellum quadruplicatum,strain BG-1

Summary Two types of filamentous mutants were derived from the unicellular blue-green alga,Agmenellum, by brief exposure to nitrosoguanidine. The parent exhibits constrictive division analogous to that of the enteric bacteria. The septate mutant exhibits septal division which is almost identical to that observed in all filamentous blue-green algae thus far described. In this mutant, the two outer wall layers fail to invaginate, leaving the daughter cells connected. The coenocytic filamentous mutant divides sporadically by both of these methods. The nuclear region of this mutant appears continuous throughout the length of the filament. It is suggested that the non-septate mutant is impaired in the coordination of cytological events leading to cell division.     

Structural analysis of human and macaque mAbs 2909 and 2.5B: implications for the configuration of the quaternary neutralizing epitope of HIV-1 gp120

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 ? in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.