Expression and purification of OsVDAC4.

The voltage-dependent anion channel (VDAC), a major component of the mitochondrial outer membrane, has emerged as an important player in cell function, survival, and death signaling. VDAC function is modulated by its interaction with proteins such as hexokinase, adenine nucleotide translocator, and apoptotic proteins like Bax. Monitoring the activity of VDAC and its modulation in the complex cellular milieu is fraught with complications. Minimizing the number of components in the study is one approach to teasing apart various aspects of its function. In this chapter, we have described detailed protocols for the purification of a rice VDAC isoform, OsVDAC4 after overexpression in a bacterial system. The protein is solubilized with LDAO and then reconstituted into liposomes or planar bilayers to verify its competence to fold into a functionally active form.

Abscisic acid flux alterations result in differential abscisic acid signaling responses and impact assimilation efficiency in barley under terminal drought stress.

Abscisic acid (ABA) is a central player in plant responses to drought stress. How variable levels of ABA under short-term versus long-term drought stress impact assimilation and growth in crops is unclear. We addressed this through comparative analysis, using two elite breeding lines of barley (Hordeum vulgare) that show senescence or stay-green phenotype under terminal drought stress and by making use of transgenic barley lines that express Arabidopsis (Arabidopsis thaliana) 9-cis-epoxycarotenoid dioxygenase (AtNCED6) coding sequence or an RNA interference (RNAi) sequence of ABA 8'-hydroxylase under the control of a drought-inducible barley promoter. The high levels of ABA and its catabolites in the senescing breeding line under long-term stress were detrimental for assimilate productivity, whereas these levels were not perturbed in the stay-green type that performed better. In transgenic barley, drought-inducible AtNCED expression afforded temporal control in ABA levels such that the ABA levels rose sooner than in wild-type plants but also subsided, unlike as in the wild type , to near-basal levels upon prolonged stress treatment due to down-regulation of endogenous HvNCED genes. Suppressing of ABA catabolism with the RNA interference approach of ABA 8'-hydroxylase caused ABA flux during the entire period of stress. These transgenic plants performed better than the wild type under stress to maintain a favorable instantaneous water use efficiency and better assimilation. Gene expression analysis, protein structural modeling, and protein-protein interaction analyses of the members of the PYRABACTIN RESISTANCE1/PYRABACTIN RESISTANCE1-LIKE/REGULATORY COMPONENT OF ABA RECEPTORS, TYPE 2C PROTEIN PHOSPHATASE Sucrose non-fermenting1-related protein kinase2, and ABA-INSENSITIVE5/ABA-responsive element binding factor family identified specific members that could potentially impact ABA metabolism and stress adaptation in barley.

Biocompatibility, absorption and safety of protein nanoparticle-based delivery of doxorubicin through oral administration in rats.

Doxorubicin, a potent anticancer drug associated with cardiotoxicity and low oral bioavailability, was loaded into apotransferrin nanoparticles to improve its pharmacological performance. Here, doxorubicin (doxo)-loaded apotransferrin nanoparticles were termed as Apodoxonano, and they were prepared by sol-oil chemistry. The pH-dependent stability of nanoparticles in simulated fluids was evaluated, and the in vitro release was investigated in phosphate-buffered saline. The pharmacokinetic and toxicity studies were conducted in Wistar rats. Nanoparticles have an average size of 75 nm, with 63% entrapment efficiency, at 10 mg w/w of apotransferrin. The particles displayed good pH-dependent stability in the pH range 1.1-7.4, but sensitive at endosomal pH of 5.5, thus facilitating intracellular drug release in endosomes. Multiplex assay showed high transport ability of nano form across epithelial cells (caco-2) when compared to doxo. Moreover, during oral administration, Apodoxonano localizes significantly in esophagus, stomach and small intestine, suggesting that it was absorbed in GI tract through epithelial lining. The drug localization was shown to be significantly lower in the heart reflecting its decreased cardiotoxic nature. The Apodoxonano with a longer bioavailability and a negligible cardiotoxicity can serve as an effective and safe vehicle of drug delivery.

Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120.

Early pregnancy associated protein-1 (Epap-1), a 90kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N=8; N=7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region.

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death.

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H2O2. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.

Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4.

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 ß-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies-spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in ß-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large ß-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.