Chicken skeletal muscle-associated macroarray for gene discovery

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Global iron-dependent gene regulation in Escherichia coli - A new mechanism for iron homeostasis

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur ( ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron- transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron- rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron- containing proteins is repressed under iron- restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of Fe-55-labeled E. coli proteins revealed a marked decrease in iron- protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron- sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Distinct subsets of hypothalamic genes are modulated by two different thermogenesis-inducing stimuli

Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-D-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.

CHANGES OF GENE EXPRESSION IN ELECTRICALLY STIMULATED AND CONTRALATERAL RAT SOLEUS MUSCLES

In this study we investigate the effect of a single session of high-intensity contractions on expression of pleiotropic genes and, in particular, those genes associated with metabolism in soleus muscle from electrically stimulated (ES) and contralateral (CL) limbs. The right limbs of male Wistar rats were submitted to contractions by 200-ms trains of electrical stimulation at 100-Hz frequency with pulses of 0.1 ms (voltage 24 3 V) delivered each second for 1 hour. Soleus muscles were isolated 1 hour after contraction, and gene expression was analyzed by a macroarray technique (Atlas Toxicology 1.2 Array; Clontech Laboratories). Electrical stimulation increased expression in 92 genes (16% of the genes present in the membrane). Sixty-six genes were upregulated in both ES and CL soleus muscles, and expression of 26 genes was upregulated in the ES muscle only. The most altered genes were those related to stress response and metabolism. Electrical stimulation also raised expression of transcription factors, translation and posttranslational modification of proteins, ribosomal proteins, and intracellular transducers/effectors/modulators. The results indicate that a single session of electrical stimulation upregulated expression of genes related to metabolism and oxidative stress in soleus muscle from both ES and CL limbs. These findings may indicate an association with tissue hypertrophy and metabolic adaptations induced by physical exercise training not only in the ES but also in the CL non-stimulated muscle, suggesting a cross-education phenomenon. Muscle Nerve 40: 838-846, 2009

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Sugarcane genes differentially expressed during water deficit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of gene expression profiles under water stress in tolerant and sensitive sugarcane plants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genes diferencialmente expressos em cana-de-açúcar inoculada com Xanthomonas albilineans, o agente causal da escaldadura da folha

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genome-Wide Analysis of Differentially Expressed Genes During the Early Stages of Tomato Infection by a Potyvirus

Plant responses against pathogens cause up-and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e. g., pathogenesis-related protein 5), regulation of the cell cycle (e. g., cytokinin-repressed proteins), signal transduction (e. g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e. g., WRKY and SCARECROW transcription factors), stress response proteins (e. g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e. g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Peanut genes identified during initial phase of Cercosporidium personatum infection

Late leaf spot (LLS), caused by the fun.-us Cercosporidium personatum, is one of the most severe diseases in peanut (Arachis hypogaea). The vast majority of commercial cultivars do not exhibit satisfactory levels of resistance to the pathogen, whereas non-commercial genotypes cv. 850 and cv. 909 are resistant to LLS and show symptoms similar to hypersensitive response (HR) lesions. In the present study, we investigated the molecular components of the initial stages of the resistance by gene expression profiling using suppression subtractive hybridization and differential screening of cDNA macroarray techniques. Gene expression analyses have allowed us to identify more than 700 peanut unique expressed sequence taus (EST) involved in several aspects of the early stages of C. personatum pathogenesis, such as components of defense signaling pathways, gene expression regulators, cell cycle controlling genes and components of the biosynthesis of transducer and antimicrobial compounds. The most significantly induced gene corresponds to a novel O'-methyltranferase, suggesting its involvement in the production of local lesions in C. personatum-resistant A. hypogea genotypes. Taken together, our results contribute to elucidate the defense strategies of peanut and provide the framework for the generation of pathogen-resistant peanut cultivars. (C) 2007 Elsevier B.V. All rights reserved.

Hypothalamic melanin-concentrating hormone is induced by cold exposure and participates in the control of energy expenditure in rats

Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)