Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Identification of Phorbol Myristate Acetate Stimulated Kinase in Zea mays

Following DEAE-Sephacel and affinity chromatography a highly enriched lipid stimulated kinase activity could be recovered with a purification fold of 1725. The peak kinase activity fraction eluted with 0.1 mM calcium from phosphatidyl serine affinity chromatography showed a major protein of 70 kD and a minor band of 55 kD molecular weight and showed kinase activity that was stimulated by phorbol myristate acetate in the presence of phosphatidylserine and calcium. The optimum requirement was 2.5 × 10^?6 M, 1.25 × 10^?4 M, 1 × 10^?4 M, and 1.7 × 10^?6 M for phorbol myristate acetate, phosphatidyl serine, oleyl acetyl glycerol and free calcium respectively. The kinase activity was inhibited by H-7 and staurosporine. The binding of [³H]-phorbol myristate acetate was associated with purified fraction as resolved by get electrophoresis and the kinase activity was also precipitated by animal protein kinase C antibodies. The present data give strong evidence for the presence of phorbol myristate acetate stimulated kinase in plants.     

Expression of an ABA-induced gene of tomato in transgenic tobacco during periods of water deficit

The gene le25 is an abscisic acid (ABA)-induced gene of tomatowhich is expressed both in wilted vegetative organs and developingseeds. Spatial and temporal expression was analysed in tobaccoplants transformed with a chimeric gene in which 5'-upstreamDNA sequences of le25 were fused to the E. coli uidA gene, whichencodes ß-glucuronidase (GUS). Histochemical stainingrevealed that GUS was expressed in all tissues of vegetativeorgans in response to water deficit. Exogenous ABA induced expressionto a lesser extent, even though ABA content was the same asdroughtstressed leaves, indicating a difference in responseto endogenous ABA compared to exogenous ABA. Water-deficit-inducedGUS expression in floral tissues was examined in pre-anthesisfloral buds from four different stages (I–IV; 11, 16,33, 49 mm bud length, respectively). While non-stressed floralorgans showed no GUS activity except in pollen at stages IIIand IV, GUS activity was water-deficit-induced in sepals ofall stages, petals of stage II, and stigmas of stage II andIII. In seeds, GUS activity was detected in both the embryoand endosperm at 15 d post-anthesis, which coincided with alarge increase in the concentration of ABA in the seed. In transgenicplants, the le25 5'-flanking DNA drove expression of GUS duringwater deficit in two modes: non-tissue-specific expression invegetative organs, and tissue-specific expression in reproductiveorgans. The location of GUS activity indicated that ABA concentrationis elevated throughout the tissues of the leaf during periodsof water deficit. Key words: Tomato, ABA, drought stress, lea gene, water deficit     

Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.     

Chiral Imidazolidin-4-one with catalytic amount of Dicationic ionic liquid act as a recoverable and reusable Organocatalyst for asymmetric Diels-Alder reaction

Imidazolidin-4-one is used as a recoverable organocatalyst for the asymmetric Diels-Alder reaction in the presence of catalytic amount of dicationic ionic liquid and trifluoroacetic acid as a co-catalyst. The Diels-Alder reaction between model substrate cyclopentadiene and crotonaldehyde gave the product in 95% conversion and 87% ee of the endo-product. The catalyst was shown better reusability when the 20 mol% of dicationic ionic liquid was used and catalyst was reused upto 5 cycles, conversion remains upto 3 recycles but ee of endo- 9 was slightly droped.     

Antifungal efficacy of Thevetia peruviana leaf extract against Alternaria solani and characterization of novel inhibitory compounds by Gas Chromatography-Mass Spectrometry analysis

Alternaria solani, a plant pathogenic fungus causes significant economical losses of potato crop. The disease is controlled primarily through some traditional methods and most commonly via the application of chemical fungicides. Fungicides treatment is not protected as chemicals pollute environment, effect health vulnerability in humans and when these harmful chemicals enter into the food chain become hazardous to all living entities. Recent efforts have focused on developing environmentally safe, long-lasting, and effective biocontrol methods for the management of plant diseases. Present research focus on screening of crude and partially purified leaf extract of Thevetia peruviana for the presence of antifungal efficacy against Alternarai solani. It was observed that 100% alcoholic crude and alcoholic fraction of partially purified extract showed maximum inhibitory activity which is due to the presence of different secondary metabolites, revealed by phytochemical screening. Active column fraction (possess best antifungal activity against Alternaria solani) was subjected to Gas Chromatography-Mass Spectrometry (GS-MS) analysis. On the basis of peaks matching of GC-MS chromatogram with available data base showed the presence of benzoic acid and oxo-benzoate in active fraction of Thevetia peruviana leaf extract which is already known chemical among the phytochemicals described for antimicrobial activity. Further research on development of herbal formulation from the same would be very helpful environment friendly approach to manage concern crop disease.     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.