Biomedical subjects

Richard R Almon

Publications and source records attributed to Richard R Almon.

17 recordsLinked to original sources

PubMed2006

Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle.

PURPOSE: The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period. METHODS: Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured. RESULTS: The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue(-1). GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue(-1), whereas GS activity fluctuates between 17 and 23 micromol min(-1) g protein(-1). Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle. CONCLUSIONS: The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS.

Algorithms↗

PubMed2005

Modeling glucocorticoid-mediated fetal lung maturation: I. Temporal patterns of corticosteroids in rat pregnancy.

Preterm birth produces neonatal respiratory distress syndrome, and dexamethasone (DEX) is administered maternally to induce fetal lung maturation in women at risk of preterm delivery. Antenatal DEX therapy is largely empirical, and administering multiple doses of DEX produces undesirable metabolic and developmental effects in the fetus. It is hypothesized that pharmacokinetic/pharmacodynamic (PK/PD) assessment of the maternal/fetal disposition and selected effects of corticosteroids will allow insight into optimal dosing methods. An optimal regimen was defined as a dosing schedule that would reproduce the endogenous prenatal steroid exposure and up-regulation of fetal lung maturational markers precociously. This report focuses on designing such a regimen from a PK standpoint in rats. The temporal profile of endogenous corticosterone in control rats was captured using a radioimmunoassay and showed that maternal and fetal corticosterone increased significantly during the last days of gestation. Six 1-mumol kg(-1) intramuscular DEX doses separated by 12-h intervals were administered maternally starting at gestational age 18, and PK was captured using a liquid chromatography-mass spectrometry assay. Unbound DEX exhibited a fetal to maternal concentration ratio of 0.55, had a free fraction of 0.2 in maternal and 0.4 in fetal plasma, and declined with a half-life of approximately 3 h. DEX PK and plasma protein binding were linear during the study, and DEX exposure caused severe adrenosuppression. These temporal steroid profiles in the fetal circulation will be used to drive the PD effects reported in a companion paper and an optimal steroid regimen will be proposed.

Animals↗

PubMed2005

Modeling glucocorticoid-mediated fetal lung maturation: II. Temporal patterns of gene expression in fetal rat lung.

Our previous report described the temporal steroid patterns during pharmacokinetic (PK) studies with dexamethasone (DEX) where doses of six 1 micromol/kg injections were given during gestational ages 18 to 20 days in rats. DEX PK was used in conjunction with the endogenous corticosterone profile to understand the regulation of fetal lung pharmacodynamics (PD). Expression of the glucocorticoid receptor (GR) and surfactant proteins A and B mRNA were chosen as lung maturational markers. GR seemed to be insensitive to the circulating glucocorticoids, indicating that unlike the adult situation, GR was not under negative feedback control of its ligand. Surfactant protein B exhibited approximately 400-fold induction in control fetal lung during the last days of gestation, and the inductive effect was even greater in the treatment group. Surfactant protein A displayed approximately 100-fold induction in control fetal lung during late gestation. However, the treatment group exhibited biphasic stimulatory and inhibitory effects for surfactant protein A. The inhibitory effect indicated that the chosen dosing scheme for DEX was not an optimal regimen. These data were used to determine by simulation the DEX regimen that would reproduce the temporal pattern of lung maturation observed in control animals. PK/PD modeling indicated that maintaining steroid exposure at approximately twice the equilibrium dissociation constant for the steroid/receptor interaction should produce optimal stimulation of both surfactant proteins. The simulations illustrate that administering smaller quantities of steroids over extended periods that produce sustained steroid exposure might be the optimal approach for designing dose-sparing antenatal corticosteroid therapy.

Animals↗

PubMed2005

Pharmacogenomic responses of rat liver to methylprednisolone: an approach to mining a rich microarray time series.

A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparations from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.

Adrenalectomy↗

PubMed2005

Corticosteroid-regulated genes in rat kidney: mining time series array data.

Kidney is a major target for adverse effects associated with corticosteroids. A microarray dataset was generated to examine changes in gene expression in rat kidney in response to methylprednisolone. Four control and 48 drug-treated animals were killed at 16 times after drug administration. Kidney RNA was used to query 52 individual Affymetrix chips, generating data for 15,967 different probe sets for each chip. Mining techniques applicable to time series data that identify drug-regulated changes in gene expression were applied. Four sequential filters eliminated probe sets that were not expressed in the tissue, not regulated by drug, or did not meet defined quality control standards. These filters eliminated 14,890 probe sets (94%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series datasets. The remaining data can then be further analyzed by clustering and mathematical modeling. Initial analysis of this filtered dataset identified a group of genes whose pattern of regulation was highly correlated with prototype corticosteroid enhanced genes. Twenty genes in this group, as well as selected genes exhibiting either downregulation or no regulation, were analyzed for 5' GRE half-sites conserved across species. In general, the results support the hypothesis that the existence of conserved DNA binding sites can serve as an important adjunct to purely analytic approaches to clustering genes into groups with common mechanisms of regulation. This dataset, as well as similar datasets on liver and muscle, are available online in a format amenable to further analysis by others.

Animals↗

PubMed2005

Temporal profiling of the transcriptional basis for the development of corticosteroid-induced insulin resistance in rat muscle.

Elevated systemic levels of glucocorticoids are causally related to peripheral insulin resistance. The pharmacological use of synthetic glucocorticoids (corticosteroids) often results in insulin resistance/type II diabetes. Skeletal muscle is responsible for close to 80% of the insulin-induced systemic disposal of glucose and is a major target for glucocorticoid-induced insulin resistance. We used Affymetrix gene chips to profile the dynamic changes in mRNA expression in rat skeletal muscle in response to a single bolus dose of the synthetic glucocorticoid methyl-prednisolone. Temporal expression profiles (analyzed on individual chips) were obtained from tissues of 48 drug-treated animals encompassing 16 time points over 72 h following drug administration along with four vehicle-treated controls. Data mining identified 653 regulated probe sets out of 8799 present on the chip. Of these 653 probe sets we identified 29, which represented 22 gene transcripts, that were associated with the development of insulin resistance. These 29 probe sets were regulated in three fundamental temporal patterns. 16 probe sets coding for 12 different genes had a profile of enhanced expression. 10 probe sets coding for eight different genes showed decreased expression and three probe sets coding for two genes showed biphasic temporal signatures. These transcripts were grouped into four general functional categories: signal transduction, transcription regulation, carbohydrate/fat metabolism, and regulation of blood flow to the muscle. The results demonstrate the polygenic nature of transcriptional changes associated with insulin resistance that can provide a temporal scaffolding for translational and post-translational data as they become available.

Animals↗

PubMed2004

Receptor/gene-mediated pharmacodynamic effects of methylprednisolone on phosphoenolpyruvate carboxykinase regulation in rat liver.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme for gluconeogenesis. To investigate underlying mechanisms of corticosteroid (CS) action in regulating glucose, temporal patterns of hepatic PEPCK gene expression, enzyme activity, and cAMP content were examined in adrenalectomized rats receiving acute and chronic methylprednisolone (MPL) treatments. After single MPL intravenous doses, PEPCK mRNA showed a fast increase, reaching a maximum at around 0.75 h, which was followed by an immediate decline to below baseline after 4 h, an apparent acute tolerance/rebound phenomenon. However, PEPCK enzyme showed continuous hyperactivity for over 72 h. This may be the result of generation of cAMP, an important inducer of PEPCK activity, which peaked at around 6 h. During 7-day subcutaneous infusion of MPL, PEPCK mRNA showed profiles consistent with single-dose results, whereas PEPCK activity increased to a comparable maximum followed by a slow decline. However, the extent of cAMP induction was markedly higher during infusion, which could be attributed to amplification of cAMP synthesis and/or a stabilizing effect of MPL on cAMP degradation. A pharmacokinetic/pharmacodynamic model was developed based on receptor/gene mechanisms of CS action. It successfully described the dual effects of MPL on regulating PEPCK message and the post-transcriptional control by cAMP. Our results exemplify the importance of the extent and duration of steroid exposure in mediating pharmacological effects. The model provides quantitation of multiple controlling factors regulating PEPCK and presents insights into its function in glucose metabolism.

Animals↗

PubMed2004

The PEPR GeneChip data warehouse, and implementation of a dynamic time series query tool (SGQT) with graphical interface.

Publicly accessible DNA databases (genome browsers) are rapidly accelerating post-genomic research (see http://www.genome.ucsc.edu/), with integrated genomic DNA, gene structure, EST/ splicing and cross-species ortholog data. DNA databases have relatively low dimensionality; the genome is a linear code that anchors all associated data. In contrast, RNA expression and protein databases need to be able to handle very high dimensional data, with time, tissue, cell type and genes, as interrelated variables. The high dimensionality of microarray expression profile data, and the lack of a standard experimental platform have complicated the development of web-accessible databases and analytical tools. We have designed and implemented a public resource of expression profile data containing 1024 human, mouse and rat Affymetrix GeneChip expression profiles, generated in the same laboratory, and subject to the same quality and procedural controls (Public Expression Profiling Resource; PEPR). Our Oracle-based PEPR data warehouse includes a novel time series query analysis tool (SGQT), enabling dynamic generation of graphs and spreadsheets showing the action of any transcript of interest over time. In this report, we demonstrate the utility of this tool using a 27 time point, in vivo muscle regeneration series. This data warehouse and associated analysis tools provides access to multidimensional microarray data through web-based interfaces, both for download of all types of raw data for independent analysis, and also for straightforward gene-based queries. Planned implementations of PEPR will include web-based remote entry of projects adhering to quality control and standard operating procedure (QC/SOP) criteria, and automated output of alternative probe set algorithms for each project (see http://microarray.cnmcresearch.org/pgadatatable.asp).

Algorithms↗

PubMed2004

The genomic response of skeletal muscle to methylprednisolone using microarrays: tailoring data mining to the structure of the pharmacogenomic time series.

High-throughput data collection using gene microarrays has great potential as a method for addressing the pharmacogenomics of complex biological systems. Similarly, mechanism-based pharmacokinetic/pharmacodynamic modeling provides a tool for formulating quantitative testable hypotheses concerning the responses of complex biological systems. As the response of such systems to drugs generally entails cascades of molecular events in time, a time series design provides the best approach to capturing the full scope of drug effects. A major problem in using microarrays for high-throughput data collection is sorting through the massive amount of data in order to identify probe sets and genes of interest. Due to its inherent redundancy, a rich time series containing many time points and multiple samples per time point allows for the use of less stringent criteria of expression, expression change and data quality for initial filtering of unwanted probe sets. The remaining probe sets can then become the focus of more intense scrutiny by other methods, including temporal clustering, functional clustering and pharmacokinetic/pharmacodynamic modeling, which provide additional ways of identifying the probes and genes of pharmacological interest.

Adrenalectomy↗

PubMed2003

Gene arrays and temporal patterns of drug response: corticosteroid effects on rat liver.

It was hypothesized that expression profiling using gene arrays can be used to distinguish temporal patterns of changes in gene expression in response to a drug in vivo, and that these patterns can be used to identify groups of genes regulated by common mechanisms. A corticosteroid, methylprednisolone (MPL), was administered intravenously to a group of 47 rats ( Rattus rattus) that were sacrificed at 17 timepoints over 72 h after MPL administration. Plasma drug concentrations and hepatic glucocorticoid receptors were measured from each animal. In addition, RNAs prepared from individual livers were used to query Affymetrix genechips for mRNA expression patterns. Statistical analyses using Affymetrix and GeneSpring software were applied to the results. Cluster analysis revealed six major temporal patterns containing 196 corticosteroid-responsive probe sets representing 153 different genes. Four clusters showed increased expression with differences in lag-time, onset rate, and/or duration of transcriptional effect. A fifth cluster showed rapid reduction persisting for 18 h. The final cluster identified showed decreased expression followed by an extended period of increased expression. These results lend new insights into the diverse hepatic genes involved in the physiologic, therapeutic, and adverse effects of corticosteroids and suggest that a limited array of control processes account for the dynamics of their pharmacogenomic effects.

Adrenal Cortex Hormones↗

PubMed2003

Modeling of corticosteroid pharmacogenomics in rat liver using gene microarrays.

Corticosteroid (CS) pharmacogenomics was studied using gene microarrays in rat liver. Methylprednisolone (MPL) was administered intravenously at 50 mg/kg. Rats were sacrificed and liver excised at 17 time points over 72 h. RNAs from individual livers were used to query Affymetrix GeneChips that contain sequences for 8000 genes. Cluster analysis revealed six temporal patterns consisting of 197 CS-responsive probes representing 143 genes. Based on our fifth-generation model of CS pharmacokinetics/pharmacodynamics (PK/PD), mechanistic models were developed to describe the time pattern for each CS-responsive gene. Two clusters showed increased expression with different effect duration. PK/PD models assuming CS stimulation of mRNA synthesis were applied. Another two clusters showed an initial decline followed by delayed increase, suggesting two mechanisms might be involved jointly. The initial suppression was captured by CS inhibition of mRNA synthesis or stimulation of degradation. CS may also stimulate the production of a biosignal (transcription factors or other hormones), which can cause secondary induction of the target mRNA. One cluster showed a very abrupt increase in message followed by rapid decrease. These genes were lymphocytic in origin and were modeled combining the fast gene induction effect of CS in lymphoid cells and its direct lymphocyte trafficking effect. Another cluster showed reduction persisting for 18 h, which was described by CS inhibition of mRNA synthesis. Our results reveal the marked diversity of genes regulated by CS via a limited array of mechanisms. These PK/PD models provide quantitation of CS pharmacogenomics and new hypotheses regarding understanding of diverse mechanisms of CS receptor-gene mediated action.

Animals↗

PubMed2003

Expression profiling and pharmacogenomics of muscle and muscle disease.

Parallel technologies are emerging for the study of DNA (single nucleotide polymorphism genotyping and association studies), and RNA (microarrays and expression profiling). Muscle tissue has many intrinsic features that make it the tissue of choice to model the application of these newly emerging technologies. Well-preserved human biopsies are readily available both from volunteers undergoing specific muscle training protocols and from neuromuscular disease patients. Furthermore, the rapid adaptation of muscle to environmental cues (e.g. exercise, conditioning and weightlessness) makes it possible to conduct time series data in humans (molecular cause and effect). Muscle is also important in drug metabolism and adverse events, and the application of single nucleotide polymorphism association studies in muscle pharmacogenomics is a field of expected future growth.

Drug Design↗

PubMed2003

In vivo multi-tissue corticosteroid microarray time series available online at Public Expression Profile Resource (PEPR).

Gene microarrays are becoming a key tool for the analysis of changes in gene expression in a variety of conditions. Use of microarrays to analyze drug responses has mainly been restricted to comparing treated versus untreated samples at a few time points. Such data do not permit the use of another important tool, pharmacokinetic/pharmacodynamic (PK/PD) modeling. Such modeling requires the simultaneous analysis of pharmacokinetic data along with time series data on dynamic responses. This report describes data obtained from two extended microarray time series (rat liver and skeletal muscle) for the in vivo responses to a single bolus dose of methylprednisolone that are uniquely available online in a single gene query format. Use of these data does not require any a priori knowledge or software normally necessary for the analysis of microarray data. Since the pharmacokinetic data and receptor model have been published, the results are amenable to PK/PD and pharmacogenomic evaluation.

Adrenal Cortex Hormones↗

PubMed2002

Quantitation of expressed message for inducible nitric oxide synthase.

Methodology for the analysis of expressed message has evolved rather rapidly since the early 1990s and will undoubtedly continue to evolve with new technology. cRNA standards are a useful tool, which allow for absolute rather than relative quantification of an expressed message. Selecting an appropriate sequence(s) for analysis has become even more important as techniques have evolved from Northern hybridization where possible multiple hybridization bands can be observed to real-time Rt-PCR approaches where only a signal is recorded. NOS2 presents a particular problem in this regard because of the high conservation of sequence with NOS1 and NOS3, as well as the existence of both multiple genes and splice variants, which may be of experimental importance.

Nitric Oxide Synthase↗

PubMed2002

Fifth-generation model for corticosteroid pharmacodynamics: application to steady-state receptor down-regulation and enzyme induction patterns during seven-day continuous infusion of methylprednisolone in rats.

A fifth-generation model for receptor/gene-mediated corticosteroid effects was proposed based on results from a 50 mg/kg i.v. bolus dose of methylprednisolone (MPL) in male adrenalectomized rats, and confirmed using data from other acute dosage regimens. Steady-state equations for receptor down-regulation and tyrosine aminotransferase (TAT) enzyme induction patterns were derived. Five groups of male Wistar rats (n = 5/group) were subcutaneously implanted with Alzet mini-pumps primed to release saline or 0.05, 0.1, 0.2, and 0.3 mg/kg/hr of MPL for 7 days. Rats were sacrificed at the end of the infusion. Plasma MPL concentrations, blood lymphocyte counts, and hepatic cytosolic free receptor density, receptor mRNA, TAT mRNA, and TAT enzyme levels were quantitated. The pronounced steroid effects were evidenced by marked losses in body weights and changes in organ weights. All four treatments caused a dose-dependent reduction in hepatic receptor levels, which correlated with the induction of TAT mRNA and TAT enzyme levels. The 7 day receptor mRNA and free receptor density correlated well with the model predicted steady-state levels. However, the extent of enzyme induction was markedly higher than that predicted by the model suggesting that the usual receptor/gene-mediated effects observed upon single/intermittent dosing of MPL may be countered by alterations in other aspects of the system. A mean IC50 of 6.1 ng/mL was estimated for the immunosuppressive effects of methylprednisolone on blood lymphocytes. The extent and duration of steroid exposure play a critical role in mediating steroid effects and advanced PK/PD models provide unique insights into controlling factors.

Adrenal Cortex Hormones↗

PubMed2002

Pharmacodynamics and pharmacogenomics of diverse receptor-mediated effects of methylprednisolone in rats using microarray analysis.

Corticosteroids such as methylprednisolone (MPL) produce many of their anti-inflammatory, immunosuppressive, and exaggerated physiological effects by receptor and gene-mediated mechanisms. The temporal pattern of change in four genes in rat tissues was measured by quantitative Northern hybridization and rtPCR after a single dose of MPL. Two profiles were observed: two genes with enhanced expression showed a slow onset and moderate rate of decline within a 24 hr time frame while two genes with reduced expression exhibited a rapid onset and prolonged suppression over a > or = 72 hr time span. These patterns are consistent with and rationalized by pharmacodynamic expectations based on earlier models. cDNA microarrays used to assess the expression levels of 5200 genes at one optimal time-point showed marked variation in baseline values. Of these, 20 genes showed statistically significant enhanced expression with increases ranging from 130 to 1690%, 31 genes exhibited reduced expression ranging from 31 to 72% of control. Many genes could be categorized as affecting acute phase/immune response, energy metabolism, microsomal metabolism, and hepatic function. These studies provide the first simultaneous assessment of the diversity in pharmacogenomic effects of corticosteroids. They also provide some insight into the advantages and limitations of microarray measurements in regard to the pharmacodynamics of drugs having complex, multi-faceted, and integrated mechanisms of action.

Animals↗

PubMed2002

Pharmacodynamics and pharmacogenomics of methylprednisolone during 7-day infusions in rats.

An array of adverse steroid effects was examined on a whole body, tissue, and molecular level. Groups of male adrenalectomized Wistar rats were subcutaneously implanted with Alzet mini-pumps giving zero-order release rates of 0, 0.1, and 0.3 mg/kg/h methylprednisolone for 7 days. The rats were sacrificed at various times during the 7-day infusion period. A two-compartment model with a zero order input could adequately describe the kinetics of methylprednisolone upon infusion. Blood lymphocyte counts dropped to a minimum by 6 h and were well characterized by the cell trafficking model. The time course of changes in body and organ (liver, spleen, thymus, gastrocnemius muscle, and lungs) weights was described using indirect response models. Markers of gene-mediated steroid effects included hepatic cytosolic free receptor density, receptor mRNA, tyrosine aminotransferase (TAT) mRNA, and TAT levels. Our fifth-generation model of acute corticosteroid pharmacodynamics was used to predict the time course of receptor/gene-mediated effects. An excellent agreement between the expected and observed receptor dynamics suggested that receptor events and mRNA autoregulation are not altered upon 7-day methylprednisolone dosing. However, the model indicated a decoupling between the receptor and TAT dynamics with this infusion. The strong tolerance seen in TAT mRNA induction could be partly accounted for by receptor down-regulation. An amplification of translation of TAT mRNA to TAT and/or a reduction in the enzyme degradation rate could account for the observed exaggerated TAT activity. Our results exemplify the importance of biological signal transduction variables in controlling receptor/gene-mediated steroid responses during chronic dosing.

Adrenalectomy↗