Intramolecular folding in human ILPR fragment with three C-rich repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br(2) footprinting experiments on a mixture of the ILPR-I3 and a 5'-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures.     

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.     

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.     

Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.     

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA₃)/ indole-3-butyric acid (IBA). BA (22.19 M) was effective for induction of multiple shoots and addition of GA₃ to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 M). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 M). However, this was associated with basal callus formation. Treatment with IBA (25–100 M) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 M for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 mol m^–2s^–1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 mol m^–2s^–1 in Q. leucotrichophora and around 1500 mol m^–2s^–1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 mol m^–2s^–1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.