Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Morphogenetic behavior of tropical marine yeast Yarrowia lipolytica in response to hydrophobic substrates

The morphogenetic behavior of a tropical marine Yarrowia lipolytica strain on hydrophobic substrates was studied. Media containing coconut oil or palm kernel oil (rich in lauric and myristic acids) prepared in distilled water or seawater at a neutral pH supported 95% of the cells to undergo a transition from the yeast form to the mycelium form. With potassium laurate, 51% of the cells were in the mycelium form, whereas with myristate, 32% were in the mycelium form. However, combinations of these two fatty acids in proportions that are present in coconut oil or palm kernel oil enhanced the mycelium formation to 65%. The culture also produced extracellular lipases during the morphogenetic change. The yeast cells were found to attach to the large droplets of the hydrophobic substrates during the transition, while the mycelia were associated with the aqueous phase. The alkane-grown yeast partitioned more efficiently in the hydrophobic phases when compared with the coconut oil-grown mycelia. A fatty acid analysis of the mycelial form revealed the presence of lauric acid in addition to the long-chain saturated and unsaturated fatty acids observed in the yeast form. The mycelia underwent a rapid transition to the yeast form with n-dodecane, a medium-chain aliphatic hydrocarbon. Thus, the fungus displayed a differential behavior towards the two types of saturated hydrophobic substrates.     

Interaction of CheY with the C-terminal peptide of CheZ

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P~CheY). The steady-state level of P~CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P~CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZ_C) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol. 359:624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZ_C peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ_C interaction. In addition, we present kinetic studies of the CheZ_C-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.     

Stages in follicle cell/oocyte interface during vitellogenesis in caecilians <Emphasis Type="Italic">Ichthyophis tricolor</Emphasis> and <Emphasis Type="Italic">Gegeneophis ramaswamii</Emphasis>: a transmission electron-microscopic study

We describe the ultrastructural organization of the vitellogenic follicle stages in two caecilian species. Monthly samples of slices of ovary of Ichthyophis tricolor and Gegeneophis ramaswamii from the Western Ghats of India were subjected to transmission electron-microscopic analysis, with special attention to the follicle cell/oocyte interface. In order to maintain uniformity of the stages among the amphibians, all the stages in the caecilian follicles were assigned to stages I–VI, the vitellogenic and post-vitellogenic follicles being assigned to stages III–VI. Stage III commences with the appearance of precursors of vitelline envelope material in the perivitelline space. Stages IV and V have been assigned appropriate substages. During the transition of stage III to stage VI oocytes, a sequential change occurs in the manifestations of follicle cells, perivitelline space, vitelline envelope and oocyte cortex. The vitelline envelope becomes a tough coat through the tunnels of which the macrovilli pass to interdigitate between the microvilli. The oocyte surface forms pinocytic vesicles that develop into coated pits and, later, coated vesicles. Contributions of the oocyte cortex to the vitelline envelope and of the follicle cells to yolk material via synthesis within them are indicated. The follicle cell/oocyte interface of vitellogenic follicles of these two caecilians resembles that in anurans and urodeles, with certain features being unique to caecilians. Thus, this paper throws light on the possible relationships of caecilians to anurans and urodeles with special reference to ovarian follicles. This research was supported by funds from the Kerala State Council for Science, Technology and Environment (KSCSTE), through the SARD facility, and by the FIST scheme of Department of Science and Technology, Government of India, New Delhi, to the Department of Zoology, University of Kerala, Thiruvananthapuram, and to the Department of Animal Science, Bharathidasan University, Thiruchirapalli (SR/FST/LSI-233/2002).     

Production of hydrogen peroxide and redox cycling can explain how sanguinarine and chelerythrine induce rapid apoptosis

Sanguinarine and chelerythrine are naturally occurring benzophenanthridines with multiple biological activities. Sanguinarine is believed to be a potential anticancer agent but its mechanism of action has not been fully elucidated. We previously found that it causes oxidative DNA damage and very rapid apoptosis that is not mediated by p53-dependent DNA damage signaling. Here we show that sanguinarine and chelerythrine cause the production of large amounts of reactive oxygen species (ROS), in particular hydrogen peroxide, which may deplete cellular antioxidants and provide a signal for rapid execution of apoptosis. Several oxidoreductases contribute to cell death induced by sanguinarine and chelerythrine which appear to be reduced upon entering the cell. We propose a model in which the generation of lethal amounts of hydrogen peroxide is explained by enzyme-catalyzed redox cycling between the reduced and oxidized forms of the phenanthridines and discuss the implications of such a mechanism for potential pharmaceutical applications.     

Combinatorial synthesis of 3-(amidoalkyl) and 3-(aminoalkyl)-2-arylindole derivatives: discovery of potent ligands for a variety of G-protein coupled receptors.

Preparation and screening of mixture libraries based on a 2-arylindole scaffold resulted in the discovery of potent ligands for a variety of G-protein coupled receptors.     

Environmental and industrial applications of Yarrowia lipolytica

Yarrowia lipolytica is a fungus that degrades hydrophobic substrates very efficiently. The fungus displays several important characteristics that have encouraged researchers to study various basic biological and biotechnological applications in detail. Although the organism has been used as model system for studying dimorphism, salt tolerance, heterologous protein expression, and lipid accumulation, there are no recent reviews on the environmental and industrial applications of this organism. Included here are applications in bioremediation of environments contaminated with aliphatic and aromatic compounds, organic pollutants, 2,4,6-trinitrotoluene, and metals. A variety of industrially important recent processes for the synthesis of β-hydroxy butyrate, l-dopa, and emulsifiers have also been reviewed. Production of unique inherent enzymes (inulinases, α-mannosidases), novel applications of esterases and lipases, and the use of the fungus for heterologous expression of biotechnologically relevant products have also been highlighted. The review while entailing a general overview focuses critically on some of the recent advances on the applications of this yeast. The examples cited here demonstrate the use of wild-type, mutant as well as genetically manipulated strains of Y. lipolytica for the development of different products, processes, and technologies. This also throws light on how a single organism can be versatile with respect to its metabolic abilities and how it can be exploited for a variety of purposes. This review will thus form a base for future developments in this field.