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Todd Smith

Publications and source records attributed to Todd Smith.

6 recordsLinked to original sources
PubMed2005

Transgene-induced RNA interference as a tool for plant functional genomics.

RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.

Arabidopsis↗
PubMed2005

Nesiritide during extracorporeal membrane oxygenation.

Nesiritide is a recombinant formulation of B-type natriuretic peptide (BNP). Preliminary experience in the adult population has shown nesiritide to be an effective agent in the treatment of decompensated congestive heart failure (CHF) in adults. Given its physiological effects, it may be an effective agent in other clinical scenarios. We report the use of nesiritide in two infants during extracorporeal membrane oxygenation (ECMO). In one patient, nesiritide in doses up to 0.09 microg.kg(-1).min(-1) were used to control mean arterial pressure while in the other patient, doses of 0.01-0.03 microg.kg(-1).min(-1) were used to augment urine output. The potential applications of nesiritide and dosing regimens for this agent in the ECMO population are discussed.

Blood Pressure↗
PubMed2004

Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems.

Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.

Animals↗
PubMed2003

Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors.

Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.

Animals↗
PubMed2002

Estrogen regulates GFAP-expression in specific subnuclei of the female rat interpeduncular nucleus: a potential role for estrogen receptor beta.

We previously demonstrated that in rat, astrocytic glial fibrillary acidic protein- (GFAP) expression in the interpeduncular nucleus (IPN) was responsive to testosterone and in females the intensity of GFAP-immunoreactivity (IR) followed the periodic hormonal changes of the estrous cycle. The aim of this study was to test whether 17beta-estradiol (E(2)), in the absence of other ovarian hormones, can influence GFAP-expression within individual subnuclei of the IPN and to determine the cellular distribution of estrogen receptor beta (ERbeta) in the IPN. Quantitative surface-density analysis was used to compare the intensity of GFAP-IR at different anterio-posterior (AP) levels of the IPN in ovariectomized female rats 24 h after treatment with E(2) or vehicle. Estrogen-treatment resulted in a significant increase in GFAP-IR in the rostrolateral subnucleus of the IPN at AP: -5.60, in the lateral-, dorsolateral-, dorsomedial- and central subnuclei at -6.04 and in the lateral subnucleus at -6.72. No significant differences were observed at -5.80 and -6.30. These results indicate that E(2), in the absence of other ovarian hormones, modulates GFAP-expression within select IPN subnuclei and that these affects are dependent on position along the AP axis. To determine whether ERbeta was a possible mediator of the observed estrogenic effects, adjacent section pairs of the IPN were immunostained for ERbeta or GFAP. Using the 'mirror' method, ERbeta-IR was detected in the cytoplasm of GFAP-immunopositive astroglia and in the nuclei of GFAP-immunonegative neurons. These findings suggest that in the IPN, E(2) may directly modulate GFAP-expression through ERbeta-mediated mechanisms.

Animals↗