Genetic engineering of indica rice with AtDREB1A gene for enhanced abiotic stress tolerance

Plant Cell, Tissue and Organ Culture (PCTOC) - Adventitious root (AR) culturing is an effective approach for obtaining bioactive compounds from the endangered plant species of Oplopanax elatus...     

A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation

Plant Cell, Tissue and Organ Culture (PCTOC) - The development of tissue-specific or inducible promoters is important for plant genetic engineering. In this study, we isolated two novel promoters...     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

Growth of AM fungi on in vitro root organ culture of Sorghum vulgare and Saccharum officinarum

Spores of Gl mosseae and Gig gigantea germinated on minimal medium produced extraradical mycelium. Gl. mosseae infected roots of S. officinarum in in vitro condition were inoculated in M medium with in vitro cultured roots of Sorghum vulgare (test roots). From the infected root of S. officinarum, the mycelium developed and it infected the test roots. The roots developed new mycelia and further the mycelia produced a few hyaline spores. In MS medium combined with soil extract, root exudate, thiamine HCl and inositol combination, spore germination and germ tube growth were higher when compared with other media.     

The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.     

Crystallographic studies on endothelial nitric oxide synthase complexed with nitric oxide and mechanism-based inhibitors

The crystal structure of the endothelial nitric oxide synthase (NOS) heme domain complexed with NO reveals close hydrogen bonding interactions between NO and the terminal guanidino nitrogen of the substrate, L-arginine. Dioxygen is expected to bind in a similar mode which will facilitate proton abstraction from L-Arg to dioxygen, a required step for O-O bond cleavage. Structures of mechanism-based NOS inhibitors, N(5)-(1-iminoethyl)-L-ornithine and N-(3-(aminomethyl)benzyl)acetamidine, provide clues on how this class of compounds operate as suicide substrate inhibitors leading to heme oxidation.     

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.     

Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin

The binding of arrestin to rhodopsin is a multistep process that begins when arrestin interacts with the phosphorylated C terminus of rhodopsin. This interaction appears to induce a conformational change in arrestin that exposes a high-affinity binding site for rhodopsin. Several studies in which synthetic peptides were used have suggested that sites on the rhodopsin cytoplasmic loops are involved in this interaction. However, the precise amino acids on rhodopsin that participate in this interaction are unknown. This study addresses the role of specific amino acids in the cytoplasmic loops of rhodopsin in binding arrestin through the use of site-directed mutagenesis and direct binding assays. A series of alanine mutants within the three cytoplasmic loops of rhodopsin were expressed in HEK-293 cells, reconstituted with 11-cis-retinal, prephosphorylated with rhodopsin kinase, and examined for their ability to bind in vitro-translated, 35S-labeled arrestin. Mutations at Asn-73 in loop I as well as at Pro-142 and Met-143 in loop II resulted in dramatic decreases in the level of arrestin binding, whereas the level of phosphorylation by rhodopsin kinase was similar to that of wild-type rhodopsin. The results indicate that these amino acids play a significant role in arrestin binding.