Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development.

The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

Seed genome hypomethylated regions are enriched in transcription factor genes.

The precise mechanisms that control gene activity during seed development remain largely unknown. Previously, we showed that several genes essential for seed development, including those encoding storage proteins, fatty acid biosynthesis enzymes, and transcriptional regulators (e.g., ABI3, FUS3) are located within hypomethylated regions of the soybean genome. These hypomethylated regions are similar to the DNA methylation valleys (DMVs), or canyons, found in mammalian cells. Here, we address the question of the extent to which DMVs are present within seed genomes and what role they might play in seed development. We scanned soybean and Arabidopsis seed genomes from postfertilization through dormancy and germination for regions that contain <5% or <0.4% bulk methylation in CG, CHG, and CHH contexts over all developmental stages. We found that DMVs represent extensive portions of seed genomes, range in size from 5-76 kb, are scattered throughout all chromosomes, and are hypomethylated throughout the plant life cycle. Significantly, DMVs are enriched greatly in transcription factor (TF) genes and other developmental genes that play critical roles in seed formation. Many DMV genes are regulated with respect to seed stage, region, and tissue, and contain H3K4me3, H3K27me3, or bivalent marks that fluctuate during development. Our results indicate that DMVs are a unique regulatory feature of both plant and animal genomes, and that a large number of seed genes are regulated in the absence of methylation changes during development, probably by the action of specific TFs and epigenetic events at the chromatin level.

Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses.

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development.

LEAFY COTYLEDON1 (LEC1), an atypical subunit of the nuclear transcription factor Y (NF-Y) CCAAT-binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene networks and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. We compared the mRNA profiles of wild-type and lec1-null mutant seeds at several stages of development to define genes that are down-regulated or up-regulated by the lec1 mutation. We used ChIP and differential gene-expression analyses in Arabidopsis seedlings overexpressing LEC1 and in developing Arabidopsis and soybean seeds to identify globally the target genes that are transcriptionally regulated by LEC1 in planta Collectively, our results show that LEC1 controls distinct gene sets at different developmental stages, including those that mediate the temporal transition between photosynthesis and chloroplast biogenesis early in seed development and seed maturation late in development. Analyses of enriched DNA sequence motifs that may act as cis-regulatory elements in the promoters of LEC1 target genes suggest that LEC1 may interact with other transcription factors to regulate distinct gene sets at different stages of seed development. Moreover, our results demonstrate strong conservation in the developmental processes and gene networks regulated by LEC1 in two dicotyledonous plants that diverged ∼92 Mya.

Equine uridine diphospho-glucuronosyltransferase 1A1, 2A1, 2B4, 2B31: cDNA cloning, expression and initial characterization of morphine metabolism.

OBJECTIVE: Uridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses. STUDY DESIGN: In vitro drug metabolism study using liver microsomes and recombinant enzyme systems. ANIMALS: Liver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons. METHODS: Based on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography-tandem mass spectrometry and enzyme kinetics determined. RESULTS: 4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide. CONCLUSIONS AND CLINICAL RELEVANCE: This is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.

Pharmacokinetics and anti-inflammatory effects of flunixin meglumine as a sole agent and in combination with phenylbutazone in exercised Thoroughbred horses.

BACKGROUND: Flunixin meglumine (FM) and phenylbutazone (PBZ) are potent anti-inflammatory agents and as such their potential to mask injuries that would otherwise keep a horse from training or racing is concerning. A common practice in racetrack medicine in the USA is to administer the two drugs within close proximity (24 hours apart) of each other, raising the concern of pharmacokinetic interactions and enhanced anti-inflammatory effects. OBJECTIVES: Describe the pharmacokinetics and effects of PBZ on the clearance of FM when administered in close proximity as well as effects on inflammatory mediators. STUDY DESIGN: Two-way randomised balanced crossover experiment. METHODS: Twelve Thoroughbred exercised horses received 500 mg FM IV alone or in combination with 2 g of IV PBZ 24 hours later. Blood and urine samples were collected prior to and for up to 120 hours post-drug administration. Whole blood samples were collected at various times and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of FM, PBZ and eicosanoids were measured using LC-MS/MS and noncompartmental pharmacokinetic analysis performed on concentration data. RESULTS: Flunixin meglumine clearance was significantly increased when horses received PBZ 24 hours post-administration (P = .03). No other differences in pharmacokinetic parameters were noted between groups. Thromboxane B2 was significantly suppressed, relative to baseline for 96 hours post-FM administration. Subsequent administration of PBZ prolonged the suppression. Prostaglandin E2 was decreased for 24 hours following administration of FM with subsequent administration of PBZ prolonging the suppression until 120 hours. PGF2alpha concentrations were decreased for up to 168 hours post-FM administration. FM administration significantly decreased 15-HETE. MAIN LIMITATIONS: Small sample size and lack of a phenylbutazone-only treatment group. CONCLUSIONS: Administration of PBZ post-FM administration increased FM clearance. The anti-inflammatory effects of FM appear to be prolonged when PBZ is administered 24 hours post-administration.

Serum concentrations, pharmacokinetic/pharmacodynamic modeling, and effects of dexamethasone on inflammatory mediators following intravenous and oral administration to exercised horses.

Corticosteroids are potent anti-inflammatory drugs and as such are commonly administered to performance and racehorses. The objectives of the current study were to describe blood and urine concentrations and the pharmacokinetics and effects on cortisol and inflammatory mediator concentrations, following intravenous and oral administration to 12 exercised horses. Horses received an intravenous administration of 40 mg of dexamethasone sodium phosphate and 20 mg of dexamethasone tablets with a 4 week washout in between administrations. Blood and urine samples were collected prior to and for up to 96 hours post drug administration. Whole blood samples were collected at various time points and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. The concentrations of dexamethasone and eicosanoids were measured using LC-MS/MS and the concentrations from both routes of administration fit simultaneously using a three-compartment pharmacokinetic model. A turnover model with inhibition of Kin gave an adequate fit to the dexamethasone-cortisol PKPD data. Serum and urine dexamethasone concentrations were at the limit of quantitation at 96 and 48 hours post administration, respectively. The volume of distribution, systemic clearance, and terminal half-life was 0.907 L/kg, 7.89 mL/h/kg, and 1.34 h, respectively. The IC50 for cortisol suppression was 0.007 ng/mL. Stimulation of dexamethasone treated blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of inflammatory biomarker production for a prolonged period of time post drug administration. The results of this study suggest that dexamethasone has a prolonged anti-inflammatory effect following intravenous or oral administration to horses.

Characterization of the in vitro CYP450 mediated metabolism of the polymorphic CYP2D6 probe drug codeine in horses.

Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, at varying substrate concentrations, was incubated with equine liver microsomes (±UDPGA) and a panel of baculovirus expressed recombinant equine P450s. Parent drug and metabolite concentrations were determined using LC-MS/MS. Incubation of codeine in equine liver microsomes generated norcodeine, morphine, codeine glucuronide and morphine 3- and 6- glucuronide. In recombinant P450 assays, the newly described CYP2D82 was responsible for catalyzing the biotransformation of codeine to morphine (Km of 247.4 µM and a Vmax of 1.6 pmol/min/pmol P450). CYP2D82 is 80% homologous to the highly polymorphic CYP2D6 enzyme, which is responsible for biotransformation of codeine to morphine in humans. CYP3A95, which shares 79% sequence homology with human CYP3A4 and CYP2D50 catalyzed the conversion of codeine to norcodeine (Km of 104.1 and 526.9 µM, Vmax of 2.8 and 2.6 pmol/min/pmol P450). In addition to describing the P450 mediated metabolism of codeine, the current study offers a candidate probe drug that could be used in vivo to study the functional implications of polymorphisms in the CYP2D gene in horses.