Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa-and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop ␤6-␣E and part of ␤7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop ␣8 and ␤-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop ␤6-␣E mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity.receptor interaction ͉ binding epitopes ͉ insect resistance ͉ mode of action

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for Nrfixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

Ethnopharmacology relevance Galphimiaglauca (Malpighiaceae) is a Mexican plant popularly used as a tranquilizer in the treatment of nervous system disorders, although it is also used to treat other common illnesses. Aim of the study The aim of this investigation is to find out if populations of Galphimiaglauca collected in different regions and ecosystems in Mexico actually belong to the same species by using the contemporary technique of DNA barcodes. Our previous metabolic profiling study demonstrates that different collections of this plant obtained from various geographical areas exhibited diverse chemical profiles in terms of the active compounds named Galphimines. We expected the DNA barcodes apart from indicating the different species of Galphimia would indicate the active populations. Materials and methods We employed matK, rpoC1 and rbcL DNA barcodes to indicate the different species. Furthermore to investigate the possible impact of the several different ecosystems where the seven populations were collected, thin layer chromatography was employed to create a partial chemical profile, which was then compared with the metabolic profiles obtained by 1H-NMR and multivariate data analysis. Results and conclusions This study showed that the seven populations here analyzed contain at least three different species of the genus Galphimia, although each individual population is homogeneous. Interestingly our TLC analysis clearly showed that the active populations displayed a distinctively unique chemical profile. This work also showed that the use of DNA barcodes combined with chemical profile analysis is an excellent approach to solve the problems of quality control in the development of Galphimia-based medicines, as well as for any breeding programs for this species.

A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.Glutamate biosynthesis can be achieved through the reductive amination of 2-oxoglutarate catalyzed by NADP+-dependent glutamate dehydrogenase (NADP+-GDH) (2). In 1970, Tempest et al. (24) demonstrated the existence of an alternative pathway for glutamate biosynthesis in Klebsiella aerogenes. This pathway comprised glutamate synthase (GOGAT) and glutamine synthetase. The function of this pathway has been demonstrated in several microorganisms (3,12,23) and in higher plants (18). Physiological studies indicate that the main role of the glutamine synthetase-GOGAT pathway is in ammonium assimilation and glutamate biosynthesis under ammonium-limited conditions (23). In Neurospora crassa, GOGAT also appears to play an important role in glutamine degradation (4). N. crassa (21), Salmonella typhimurium (16), and Escherichia coli (5, 19) mutants lacking GOGAT activity have been obtained, and their characterization has contributed to the understanding of the role of GOGAT in glutamate biosynthesis.Although GOGAT activity has been previously detected in Saccharomyces cerevisiae (22), the role of GOGAT in glutamate biosynthesis and its regulation are poorly understood. Mutants altered in NADP+-GDH have been isolated from S. cerevisiae (7). The isolation of GOGAT-less mutants has been reported elsewhere (26); however, their genetic and physiological characterization has not been published, so the physiological function of GOGAT still remains obscure. Our results indicate that GOGAT plays a role in glutamate biosynthesis under conditions of ammonium excess and that under ammonium-limited conditions, both NADP+-GDH and GOGAT can participate in the synthesis of glutamate. We also present evidence suggesting that S. cerevisiae has two GOGAT activities.MATERIALS AND METHODS Strains. Table 1 describes the characteristics of the different strains used in this study.Growth conditions. Strains were routinely grown on minimal medium (MM) containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco * Corresponding author.Laboratories, Detroit, Mich.). Glucose (2% [wt/vol]) was used as the carbon source, and 40 mM (NH4)2SO4 was used as the nitrogen source. Amino acids needed to satisfy auxotrophic requirements were added at 0.01% (wt/vol). Cells wer...