RESUMO
BACKGROUND: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3). OBJECTIVES: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses. METHODS: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species. RESULTS: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen. CONCLUSIONS: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.
Assuntos
Disruptores Endócrinos/toxicidade , Útero/efeitos dos fármacos , Animais , Biomarcadores/análise , Diarileptanoides/toxicidade , Estrogênios/toxicidade , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Fitoestrógenos/toxicidade , Útero/metabolismoRESUMO
We report here the transcriptional responses in Saccharomyces cerevisiae to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2. Deleting RNH201 alters RNA expression of 349 genes by ≥1.5-fold (q-value <0.01), of which 123 are upregulated and 226 are downregulated. Differentially expressed genes (DEGs) include those involved in stress responses and genome maintenance, consistent with a role for RNase H2 in removing ribonucleotides incorporated into DNA during replication. Upregulated genes include several that encode subunits of RNA polymerases I and III, and genes involved in ribosomal RNA processing, ribosomal biogenesis and tRNA modification and processing, supporting a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA genes. A role in R-loop resolution is further suggested by a higher average GC-content proximal to the transcription start site of downregulated as compared to upregulated genes. Several DEGs are involved in telomere maintenance, supporting a role for RNase H2 in resolving RNA-DNA hybrids formed at telomeres. A large number of DEGs encode nucleases, helicases and genes involved in response to dsRNA viruses, observations that could be relevant to the nucleic acid species that elicit an innate immune response in RNase H2-defective humans.
Assuntos
Reparo do DNA , Regulação Fúngica da Expressão Gênica , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Composição de Bases , Domínio Catalítico , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Redes Reguladoras de Genes , Genes de RNAr , Instabilidade Genômica , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Telômero/genética , Telômero/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
BACKGROUND: The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software. RESULTS: The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1 was significantly upregulated in steatotic liver following I/R. CONCLUSION: Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
Assuntos
Fígado Gorduroso/metabolismo , Chaperonas Moleculares/metabolismo , Proteômica , Traumatismo por Reperfusão/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Regulação para Baixo , Eletroforese em Gel Bidimensional , Estresse do Retículo Endoplasmático , Fígado Gorduroso/patologia , Masculino , Espectrometria de Massas , Proteoma/metabolismo , Ratos , Ratos Zucker , Regulação para Cima , Isquemia QuenteRESUMO
The era of toxicogenomics has introduced a new way of monitoring the effect of environmental stressors and toxicants on biological systems via quantification of changes in gene expression. Because the liver is one of the major organs for synthesis and secretion of substances which metabolize endogenous and exogenous materials, there has been a great deal of interest in elucidating predictive and mechanistic genomic markers of hepatotoxicity. This mini-review will bring context to a limited number of toxicogenomics studies which used genomics to evaluate the transcriptional changes in blood and liver in response to acetaminophen (APAP) or other liver toxicants, but differed according to the classification of interest (COI), i.e., the partitioning of the samples a priori according to a common toxicological characteristic. The toxicogenomics studies highlighted are characterized by a classification of either no/low vs. high APAP dose exposure, none vs. observed necrosis, and severity of necrosis. The overlap or lack thereof between the gene classifiers and the modulated biological processes that are elucidated will be discussed to enhance the understanding of the effect of the particular COI model and experimental design used for prediction.
RESUMO
Gene expression array technology has reached the stage of being routinely used to study clinical samples in search of diagnostic and prognostic biomarkers. Due to the nature of array experiments, which examine the expression of tens of thousands of genes simultaneously, the number of null hypotheses is large. Hence, multiple testing correction is often necessary to control the number of false positives. However, multiple testing correction can lead to low statistical power in detecting genes that are truly differentially expressed. Filtering out non-informative genes allows for reduction in the number of null hypotheses. While several filtering methods have been suggested, the appropriate way to perform filtering is still debatable. We propose a new filtering strategy for Affymetrix GeneChips®, based on principal component analysis of probe-level gene expression data. Using a wholly defined spike-in data set and one from a diabetes study, we show that filtering by the proportion of variation accounted for by the first principal component (PVAC) provides increased sensitivity in detecting truly differentially expressed genes while controlling false discoveries. We demonstrate that PVAC exhibits equal or better performance than several widely used filtering methods. Furthermore, a data-driven approach that guides the selection of the filtering threshold value is also proposed.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Componente Principal , Animais , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , RatosRESUMO
Heart muscle is metabolically versatile, converting energy stored in fatty acids, glucose, lactate, amino acids, and ketone bodies. Here, we use mouse models in ketotic nutritional states (24 h of fasting and a very low carbohydrate ketogenic diet) to demonstrate that heart muscle engages a metabolic response that limits ketone body utilization. Pathway reconstruction from microarray data sets, gene expression analysis, protein immunoblotting, and immunohistochemical analysis of myocardial tissue from nutritionally modified mouse models reveal that ketotic states promote transcriptional suppression of the key ketolytic enzyme, succinyl-CoA:3-oxoacid CoA transferase (SCOT; encoded by Oxct1), as well as peroxisome proliferator-activated receptor alpha-dependent induction of the key ketogenic enzyme HMGCS2. Consistent with reduction of SCOT, NMR profiling demonstrates that maintenance on a ketogenic diet causes a 25% reduction of myocardial (13)C enrichment of glutamate when (13)C-labeled ketone bodies are delivered in vivo or ex vivo, indicating reduced procession of ketones through oxidative metabolism. Accordingly, unmetabolized substrate concentrations are higher within the hearts of ketogenic diet-fed mice challenged with ketones compared with those of chow-fed controls. Furthermore, reduced ketone body oxidation correlates with failure of ketone bodies to inhibit fatty acid oxidation. These results indicate that ketotic nutrient environments engage mechanisms that curtail ketolytic capacity, controlling the utilization of ketone bodies in ketotic states.
Assuntos
Miocárdio/metabolismo , Animais , Isótopos de Carbono/química , Coenzima A-Transferases/metabolismo , Imuno-Histoquímica/métodos , Corpos Cetônicos/química , Cetonas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Miócitos Cardíacos/citologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , RatosRESUMO
DNA microarray studies offer a robust method for nonbiased analysis of whole genome messenger ribonucleic acid expression patterns. A growing number of studies have applied this experimental approach to studies on ethanol either in cell culture of animal models of ethanol exposure or self-administration. Expression profiling has identified novel gene networks responding to ethanol or differing across animal strains with differing responses to ethanol. Recent studies have shown benefit for meta-analysis of microarray data across different laboratories. Gene network analysis offers unique opportunities for understanding the molecular mechanisms of ethanol responses, toxicity and addiction. Eventually, such work may generate novel targets for future pharmacotherapy. To fully capitalize on the prom ise alluded to above, particularly in regard to meta-analysis of microarray data, it is critical that high quality standards are followed in the generation and analysis of microarray studies. This chapter will discuss experience of our laboratory in performing and analyzing microarray studies on ethanol, focusing discussion mainly on short oligonucleotide microarrays (Affymetrix). However, the general principals of technique and analysis that are discussed have broad applicability to other types of microarray platforms and experimental designs.
Assuntos
Encéfalo/efeitos dos fármacos , Etanol/toxicidade , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Células Cultivadas , Biologia Computacional , Interpretação Estatística de Dados , Perfilação da Expressão Gênica/normas , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Modelos Animais , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos/normas , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Controle de Qualidade , RNA Mensageiro/análise , Reprodutibilidade dos Testes , Projetos de PesquisaRESUMO
This article summarizes the proceedings of a symposium that took place at the 2005 meeting of the Research Society on Alcoholism. The organizers/chairs were Daniel Goldowitz and Katheen A. Grant. The presentations were as follows: (1) High-Throughput Screening for Ethanol Phenotypes, by Douglas B. Matthews and Kristin M. Hamre; (2) Genetic Basis of Schedule-Induced Polydipsia in Mice, by Guy Mittleman and Elissa J. Chesler; (3) Effects of Stress and Ethanol Dependence on Ethanol Self-administration in Inbred and Mutant Mice, by Howard C. Becker and Marcelo F. Lopez; (4) Changes in Dopaminergic Mechanisms Associated With Ethanol Dependence, by Sara R. Jones and Tiffany A. Mathews; and (5) Defining Brain Region-Specific Gene Networks Relevant to Ethanol Behaviors, by Michael F. Miles and Robnet Kerns.
Assuntos
Alcoolismo/genética , Ansiedade/genética , Camundongos Endogâmicos , Estresse Fisiológico/genética , Animais , Comportamento Animal , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Dopamina/fisiologia , Meio Ambiente , Etanol/administração & dosagem , Camundongos , Camundongos Mutantes , Fenótipo , AutoadministraçãoRESUMO
SUMMARY: SScore is an R package that facilitates the comparison of gene expression between Affymetrix GeneChips using the S-score algorithm. The S-score algorithm uses probe level data directly to assess differences in gene expression, without requiring a preliminary separate step of probe set expression summary estimation. Therefore, the algorithm avoids introduction of error associated with the expression summary estimation process and has been demonstrated to improve the accuracy of identifying differentially expressed genes. The S-score produces accurate results even when few or no replicates are available. AVAILABILITY: The R package SScore is available from Bioconductor at http://www.bioconductor.org
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Algoritmos , Inteligência Artificial , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reconhecimento Automatizado de Padrão/métodos , Linguagens de Programação , Software , Interface Usuário-ComputadorRESUMO
This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.
Assuntos
Etanol/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica , Alcoolismo/genética , Animais , Meio Ambiente , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Locos de Características Quantitativas , Sono/genética , Especificidade da EspécieRESUMO
MUC1 is expressed at the cell surface of epithelial cancers. We have shown previously that MUC1 conjugated to keyhole limpet hemocyanin (KLH) plus the saponin immunological adjuvant QS-21 induces consistent high titer IgM and IgG antibodies in patients after treatment of their primary or metastatic cancers. KLH however is poorly soluble and heterogeneous making it difficult to work with, and we hypothesize that changing carrier proteins mid-way through a vaccination schedule would further increase antibody titers. Consequently, there is need for an alternative potent carrier protein. Duck Hepatitis B core antigen (DHBcAg) has a molecular weight of approximately 25kDa and is easily purified as a single band, but it self aggregates into particles of approximately 6.4x10(6)Da. Consequently, it is highly immunogenic, easy to work with and amenable to chemical and genetic conjugation to antigens such as MUC1. We compare here in mice the immunogenicity of MUC1 chemically conjugated to KLH or DHBcAg and MUC1-DHBcAg recombinant protein after an initial series of three vaccinations and then after an additional series of three vaccinations with the same or opposite carrier, all mixed with the saponin immunological adjuvant GPI-0100. High titer IgG antibodies were observed in all groups after the initial three vaccinations: MUC1-DHBcAg median ELISA titer 1/51200, RecMUC1-DHBcAg 1/25600 and MUC1-KLH 1/12800. This increased to 1/6553600 after the second set of three immunizations when the carrier remained the same in all three groups, but titers were significantly lower when the carriers were changed for the final three immunizations. These data demonstrate that DHBcAg is an excellent carrier protein and that changing carrier proteins does not further augment immunogenicity.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/imunologia , Hemocianinas/administração & dosagem , Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Vírus da Hepatite B do Pato/imunologia , Mucina-1/imunologia , Sequência de Aminoácidos , Animais , Proteínas do Sistema Complemento/fisiologia , Citotoxicidade Imunológica , Feminino , Hemocianinas/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Imunização , Camundongos , Dados de Sequência Molecular , Vacinas Conjugadas/imunologiaRESUMO
Activation of the mesolimbic dopamine reward pathway by acute ethanol produces reinforcement and changes in gene expression that appear to be crucial to the molecular basis for adaptive behaviors and addiction. The inbred mouse strains DBA/2J and C57BL/6J exhibit contrasting acute behavioral responses to ethanol. We used oligonucleotide microarrays and bioinformatics methods to characterize patterns of gene expression in three brain regions of the mesolimbic reward pathway of these strains. Expression profiling included examination of both differences in gene expression 4 h after saline injection or acute ethanol (2 g/kg). Using a rigorous stepwise method for microarray analysis, we identified 788 genes differentially expressed in control DBA/2J versus C57BL/6J mice and 307 ethanol-regulated genes in the nucleus accumbens, prefrontal cortex, and ventral tegmental area. There were strikingly divergent patterns of ethanol-responsive gene expression in the two strains. Ethanol-responsive genes also showed clustering at discrete chromosomal regions, suggesting local chromatin effects in regulation. Ethanol-regulated genes were generally related to neuroplasticity, but regulation of discrete functional groups and pathways was brain region specific: glucocorticoid signaling, neurogenesis, and myelination in the prefrontal cortex; neuropeptide signaling and developmental genes, including factor Bdnf, in the nucleus accumbens; and retinoic acid signaling in the ventral tegmental area. Bioinformatics analysis identified several potential candidate genes for quantitative trait loci linked to ethanol behaviors, further supporting a role for expression profiling in identifying genes for complex traits. Brain region-specific changes in signaling and neuronal plasticity may be critical components in development of lasting ethanol behavioral phenotypes such as dependence, sensitization, and craving.
Assuntos
Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting/métodos , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Esquema de Medicação , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Análise em Microsséries/métodos , Análise Multivariada , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Locos de Características Quantitativas/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da Espécie , Área Tegmentar Ventral/metabolismoRESUMO
This article summarizes the proceedings of a symposium presented at the 2004 annual meeting of the Research Society on Alcoholism in Vancouver, British Columbia, Canada. The organizers and chairs were William J. McBride and Michael F. Miles. The presentations were (1) Molecular Triangulation on Gene Expression Patterns in Behavioral Responses to Acute Ethanol, by Robnet T. Kerns; (2) Gene Expression in Limbic Regions After Ethanol Self-Infusion Into the Posterior Ventral Tegmental Area, by Zachary A. Rodd; (3) Microarray Analysis of CNS Limbic Regions of Inbred Alcohol-Preferring and -Nonpreferring rats and Effects of Alcohol Drinking, by Wendy N. Strother and Howard J. Edenberg; and (4) Microarray Analysis of Mouse Lines Selected for Chronic Ethanol Withdrawal Severity: The Convergence of Basal, Ethanol Regulated, and Proximity to Ethanol Quantitative Trait Loci to Identify Candidate Genes, by Joel G. Hashimoto and Kristine M. Wiren.
Assuntos
Alcoolismo/genética , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Regulação da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Locos de Características Quantitativas , Índice de Gravidade de Doença , Síndrome de Abstinência a Substâncias/genéticaRESUMO
In the past several years, oligonucleotide microarrays have emerged as a widely used tool for the simultaneous, non-biased measurement of expression levels for thousands of genes. Several challenges exist in successfully utilizing this biotechnology; principal among these is analysis of microarray data. An experiment to measure differential gene expression can consist of a dozen microarrays, each consisting of over a hundred thousand data points. Previously, we have described the use of a novel algorithm for analyzing oligonucleotide microarrays and assessing changes in gene expression. This algorithm describes changes in expression in terms of the statistical significance (S-score) of change, which combines signals detected by multiple probe pairs according to an error model characteristic of oligonucleotide arrays. Software is available that simplifies the use of the application of this algorithm so that it may be applied to improving the analysis of oligonucleotide microarray data. The application of this method to problems of the central nervous system is discussed.