Visualization of protein kinases in lymphocytes stimulated to proliferate with concanavalin A or inhibited with a phorbol ester

Stimulation of lymphocytes with a mitogenic lectin such as concanavalin A (ConA) results in differentiation and cell division. Among the changes which occur after stimulation are increases in phosphorylation of proteins and in protein kinase activity. We used a high-resolution, nondenaturing gel system to separate and visualize protein kinases in situ. We have clearly identified both autophosphorylating and substrate-dependent kinases. One band of cyclic AMP-dependent kinase activity was significantly enhanced in lectin-stimulated cells. In contrast, treatment of the cells with phorbol ester under conditions which depress stimulation caused a decrease in the activity of one kinase.     

Quod erat demonstrandum? The mystery of experimental validation of apparently erroneous computational analyses of protein sequences

Background  

Computational predictions are critical for directing the experimental study of protein functions. Therefore it is paradoxical when an apparently erroneous computational prediction seems to be supported by experiment.     

Improved Nutrient Use Efficiency Increases Plant Growth of Rice with the Use of IAA-Overproducing Strains of Endophytic Burkholderia cepacia Strain RRE25

Effect of indole acetic acid (IAA)-overproducing mutants of Burkholderia cepacia (RRE25), a member of β-subclass of Proteobacteria and naturally occurring rice endophyte, was observed on the growth of rice (Oryza sativa L.) plants grown under greenhouse conditions. Nine mutants were characterized for altered biosynthesis of IAA after nitrous acid mutagenesis. These mutants were grouped into two classes: class I mutants have reduced production of IAA as compared to the wild type, while class II mutants showed overproduction of IAA. Mutants of both classes and RRE25, the parent (wild type), were inoculated on rice seedlings of two cultivars (Sarjoo-52 and NDR-97). Uptake of nitrogen, phosphorous, and potassium was estimated in these plants. Significant increase in the amount of uptake of all three elements was observed when inoculated with the IAA-overproducing mutants over control as well as in the plants inoculated with the wild type (RRE25). Effect of inoculation of IAA-overproducing mutants was more pronounced on the uptake of phosphorous in cultivar NDR-97 than Sarjoo-52, while it was opposite with respect to potassium uptake. Any significant difference was not observed in nitrogen uptake among the two cultivars. It shows that the host also plays an important role in the beneficial endophytic association. It was concluded from these results that one of the possible mechanisms of growth promotion of rice plants inoculated with bacterial endophytes is their effects on an increase in the capability of nutritional uptake possible through the effect of IAA production which results in proliferation of root system that could mine more nutrients from the soil.     

Galactose metabolism of mammalian erythrocytes

Galactose metabolism in the red cells shows marked interspecies and even intraspecies variations. Red cells of guinea pig, dog and a group of rabbits metabolize galactose to a higher extent than those of other species, including human. In the rabbit, the difference in red cell galactokinase activity could not be correlated to the overall utilization of this sugar in the body.     

Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms

Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (K_d = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications.