AmpliTaq and AmpliTaq Gold DNA Polymerase The Most Referenced Brand of DNA Polymerase in the World Date: 2005-05 Notes: Authors are listed alphabetically J. Biol. Chem. (223) Ezoe, S., I. Matsumura, et al. (2005). "GATA Transcription Factors Inhibit Cytokine-dependent Growth and Survival of a Hematopoietic Cell Line through the Inhibition of STAT3 Activity." J. Biol. Chem. 280(13): 13163-13170. http://www.jbc.org/cgi/content/abstract/280/13/13163 Although GATA-1 and GATA-2 were shown to be essential for the development of hematopoietic cells by gene targeting experiments, they were also reported to inhibit the growth of hematopoietic cells. Therefore, in this study, we examined the effects of GATA-1 and GATA-2 on cytokine signals. A tamoxifen-inducible form of GATA-1 (GATA-1/ERT) showed a minor inhibitory effect on interleukin-3 (IL-3)-dependent growth of an IL-3-dependent cell line Ba/F3. On the other hand, it drastically inhibited TPO-dependent growth and gp130-mediated growth/survival of Ba/F3. Similarly, an estradiol-inducible form of GATA-2 (GATA-2/ER) disrupted thrombopoietin (TPO)-dependent growth and gp130-mediated growth/survival of Ba/F3. As for this mechanism, we found that both GATA-1 and GATA-2 directly bound to STAT3 both in vitro and in vivo and inhibited its DNA-binding activity in gel shift assays and chromatin immunoprecipitation assays, whereas they hardly affected STAT5 activity. In addition, endogenous GATA-1 was found to interact with STAT3 in normal megakaryocytes, suggesting that GATA-1 may inhibit STAT3 activity in normal hematopoietic cells. Furthermore, we found that GATA-1 suppressed STAT3 activity through its N-zinc finger domain. Together, these results suggest that, besides the roles as transcription factors, GATA family proteins modulate cytokine signals through protein-protein interactions, thereby regulating the growth and survival of hematopoietic cells. Maxwell, M. A., M. E. Cleasby, et al. (2005). "Nur77 Regulates Lipolysis in Skeletal Muscle Cells: EVIDENCE FOR CROSS-TALK BETWEEN THE {beta}-ADRENERGIC AND AN ORPHAN NUCLEAR HORMONE RECEPTOR PATHWAY." J. Biol. Chem. 280(13): 12573-12584. http://www.jbc.org/cgi/content/abstract/280/13/12573 Skeletal muscle is a major mass peripheral tissue that accounts for [~]40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces {beta}-adrenergic receptor ({beta}-AR)-mediated adaptive thermogenesis. {beta}-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30-60 min of {beta}-AR agonist (isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (>100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP- activated protein kinase {gamma}3, UCP3, CD36, adiponectin receptor 2, GLUT4, and caveolin- 3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross- talk between the nuclear hormone receptor and {beta}-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity. Agorio, A., C. Chalar, et al. (2003). "Alternative mRNAs Arising from Trans-splicing Code for Mitochondrial and Cytosolic Variants of Echinococcus granulosus Thioredoxin Glutathione Reductase." J. Biol. Chem. 278(15): 12920-12928. http://www.jbc.org/cgi/content/abstract/278/15/12920 Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans- spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin. Kaneto, H., A. Sharma, et al. (2002). "Induction of c-Myc Expression Suppresses Insulin Gene Transcription by Inhibiting NeuroD/BETA2-mediated Transcriptional Activation." J. Biol. Chem. 277(15): 12998-13006. http://www.jbc.org/cgi/content/abstract/277/15/12998 Insulin biosynthesis and secretion are critical for pancreatic [beta]-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in [beta]-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel- shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in [beta]TC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in [beta]-cells suppresses the insulin gene transcription by inhibiting NeuroD- mediated transcriptional activation. This mechanism may explain some of the [beta]-cell dysfunction found in diabetes. Chiang, M.-C., Y.-C. Lee, et al. (2005). "cAMP-response Element-binding Protein Contributes to Suppression of the A2A Adenosine Receptor Promoter by Mutant Huntingtin with Expanded Polyglutamine Residues." J. Biol. Chem. 280(14): 14331-14340. http://www.jbc.org/cgi/content/abstract/280/14/14331 Huntington's disease is a neurodegenerative disease resulting from a CAG (glutamine) trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The role of the striatum-enriched A2A adenosine receptor (A2A-R) in Huntington's disease has attracted much attention lately. In the present study, we found that expression of mutant Htt with expanded poly(Q) significantly reduced the transcript levels of the endogenous A2A-R in PC12 cells and primary striatal neurons. Cotransfection of various promoter constructs of the A2A-R gene and an expression construct of poly(Q)-expanded Htt revealed that the Htt mutant suppressed the core promoter activity of the A2A-R gene. Stimulation of the A2A-R using CGS21680 forskolin, and a constitutively active cAMP-response element-binding protein (CREB) mutant elevated the reduced promoter activity of the A2A-R gene by mutant Htt. Moreover, the effect of CGS was blocked by an A2A-R-selective antagonist (CSC), two inhibitors of protein kinase A, and two dominant negative mutants of (CREB). The protein kinase A/CREB pathway therefore is involved in regulating A2A-R promoter activity. Consistently, an atypical CRE site (TCCAGG) is located in the core promoter region of the A2A-R gene. Electrophoretic gel mobility shift assay and mutational inactivation further demonstrated the functional binding of CREB to the core promoter region and showed that expression of poly(Q)-expanded Htt abolished the binding of CREB to this site. Stimulation of the A2A-R restored the reduced CREB binding caused by the mutant and concurrently reduced mutant Htt aggregation. Collectively, the poly(Q)-expanded mutant Htt suppressed expression of the