Biochemical characterization and crystallization of recombinant 3-phosphoglycerate kinase of Plasmodium falciparum

Human malaria parasite Plasmodium falciparum depends largely on glycolytic pathway for energy metabolism during the intraerythrocytic life stage. Therefore, enzymes of the glycolytic pathway could offer potential drug targets provided novel biochemical and/or structural features of the parasitic enzymes, which distinguish them from the host counterpart, could be identified. 3-Phosphoglycerate kinase (EC 2.7.2.3) catalyzes an important phosphorylation step leading to the production of ATP in the glycolytic pathway. We have expressed recombinant 3-phosphoglycerate kinase of P. falciparum in Escherichia coli. The recombinant protein purified from the soluble fraction of E. coli is enzymatically active. The apparent K(m) values determined for adenosine triphosphate (ATP) and 3-phosphoglycerate (3-PGA) are 0.63 and 0.52 mM, respectively. The enzyme activity was temperature-sensitive. Suramin was found to inhibit the recombinant enzyme with an IC(50) value of 7 microM. We have crystallized the enzyme form in hexagonal space group P6(1)22 (or its enantiomorphic space group) with unit cell parameters a=b=130.7, c=263.9 A. Native data have been collected at 3.0-A resolution.     

The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.     

Pollen anomaly - a clue to natural hybridity in Argemone (Papaveraceae)

Studies in the pollen morphology of Argemone mexicana (a diploid species, 2n=28), A. ochroleuca (a tetraploid, 2n=56), the wild populations of which growing side by side, and of a triploid (2n=42) observed in the population of A. ochroleuca, have been carried out using light and scanning electron microscope. Pollen grains in both the species are 3 (-4)-zonocolpate with reticulate exine surface, and are distinguishable only on the basis of pollen size, the tetraploid species having larger pollen grains. In the triploid plant, a large number of grains deviates from the normal 3-zonocolpate condition showing a number of apertural types, such as 1-aperturate-operculate, 2-colpate, 3-syncolpate, 4-zonocolpate and spiraperturate. Besides, planaperturate 3-colpate-trilobed grains also occur. The triploid is also characterised by 'minigrains' (size 13-19 μm), a feature not observed in A. mexicana and A. ochroleuca. Since these species are reported to be genetically close, the occurrence of the triploids (forming nearly 5% of the population of A. ochroleuca) indicates the possibility of a natural hybridization between A. ochroleuca and A. mexicana, which is well pronounced on its anomalous pollen features serving as marker for such a hybrid occurring in nature.     

Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata

Vigna radiata or mungbean belongs to the legume family of plants. Mature mungbean seeds are rich source of dietary proteins for human nutrition. The present study was aimed to analyze the comparative protein profiles of two cotyledon types, Cot and Cot E, prior to and during early time points of shoot morphogenic induction to understand the unique differential regeneration response in these two explant types which was reported earlier. These explants were grown separately in vitro on the shoot induction medium (SIM) containing Gamborg’s B5 basal nutrient composition supplemented with 15 μM N₆-benzyladenine. Isolation and characterization of the proteomes from Cot and Cot E explants at different time points, during early events of shoot differentiation, were performed using two dimensional gel electrophoresis following matrix assisted laser desorption-ionization tandem mass spectrometry. A total of 112 differentially identified proteins were classified according to their putative biological function. The differential control of protein synthesis between these explants under control condition, i.e. before in vitro culture, was also noted. In Cot E explants SIM induced prompt acquisition of competence for direct shoot morphogenesis probably through fast phytohormone signaling. Over accumulated proteins in Cot E indicated stimulation of several metabolic and associated pathways earlier than Cot explants. Abundance of stress and defense related proteins in Cot E explants was presumably to cope up with stressful cultural condition. Enhanced accumulation of folding-assisted proteins involved in organogenesis mediated cellular reprogramming in Cot E explants contributed further in rapid and efficient regeneration responsiveness.     

Small molecule modulators of eukaryotic initiation factor 2α kinases,the key regulators of protein synthesis

Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.     

Amberlite IR-120H catalyzed MCR: Design,synthesis and crystal structure analysis of 1,8-dioxodecahydroacridines as potential inhibitors of sirtuins

A rapid, inexpensive and high yielding method has been developed for the synthesis of 1,8-dioxodecahydroacridines using Amberlite IR-120H as a reusable catalyst under open air. These compounds were designed as potential inhibitors of sirtuins and prepared via the MCR of 5,5-dimethyl-1,3-cyclohexanedione, (hetero)aryl aldehydes and (hetero)aromatic amines under mild conditions. Further structure elaboration of a representative compound was performed via Pd catalyzed C–C bond forming reactions. The crystal structure analysis and H-bonding patterns along with in vitro inhibitory activity against yeast Sir2 of the same compound is presented. Docking studies indicated that the compound interacts well with the yeast Sir2.     

Yield, growth and physiological responses of mung bean [Vigna radiata (L.) Wilczek] genotypes to waterlogging at vegetative stage

A study was conducted to examine the physiological response of contrasting mung bean (Vigna radiata) genotypes viz., T 44 & MH–96–1 (tolerant) and Pusa Baisakhi & MH–1K–24 (sensitive) under waterlogging conditions. Plants were waterlogged at vegetative stage (30 days after sowing) for 3, 6 and 9 days. Waterlogging resulted in decreased leaf area, crop growth rate, root growth and nodules number, membrane stability index, photosynthesis rate, chlorophyll and carotenoid contents, flowering rate, pod setting, yield and altered dry matter partitioning. Sensitive genotypes showed large reductions in aforementioned physiological traits and slow recovery in photosynthesis rate. On the other hand, tolerant genotypes maintained higher photosynthetic rate, chlorophylls and carotenoids, growth rate, membrane stability and fast photosynthetic recovery under waterlogging. After 9 days of exposure to waterlogging, photosynthetic rate and yield losses in most sensitive genotype (MH-1K-24) were 83 and 85 %, respectively. On an average, photosynthetic loss at 3, 6 and 9 days of waterlogging was 43, 51, and 63 %, respectively, while grain yield loss was 20, 34 and 52 % respectively.