5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has previously been reported to be taken up into cells and phosphorylated to form ZMP, an analog of 5′-AMP. This study was designed to determine whether AICAR can activate AMP-activated protein kinase (AMPK) in skeletal muscle with consequent phosphorylation of acetyl-CoA carboxylase (ACC), decrease in malonyl-CoA, and increase in fatty acid oxidation. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red blood cells, 200 μU/ml insulin, and 10 mM glucose with or without AICAR (0.5–2.0 mM). Perfusion with medium containing AICAR was found to activate AMPK in skeletal muscle, inactivate ACC, and decrease malonyl-CoA. Hindlimbs perfused with 2 mM AICAR for 45 min exhibited a 2.8-fold increase in fatty acid oxidation and a significant increase in glucose uptake. No difference was observed in oxygen uptake in AICAR vs. control hindlimb. These results provide evidence that decreases in muscle content of malonyl-CoA can increase the rate of fatty acid oxidation.

The relationship between dNTP levels and DNA synthesis was investigated using ␣ factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G 1 /S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G 1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37°C before being released to 25°C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G 1 levels. Accumulation of dNTPs at G 1 /S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25°C were able to replicate when subsequently switched to 37°C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold. Hydroxyurea (HU)1 is a potent inhibitor of the enzyme ribonucleotide reductase (RNR) and inhibits DNA replication in a wide variety of cells, including Saccharomyces cerevisiae (1). The simplest explanation for HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. HU treatment has been shown to reduce the purine dNTP pools in a variety of mammalian cells (2-7); however, conflicting data exist concerning its modulation of pyrimidine dNTP pool levels. Furthermore, even for purine dNTPs, HU has only rarely been shown to cause a complete depletion of the dGTP or dATP pools (2-4). More commonly, HU results in only partial depletion of the purine dNTP pools (5-7). The complicated, often reciprocal, changes in individual dNTP pools that occur in HU-treated mammalian cells may be due to the compensatory activities of deoxyribonucleotide salvage pathways in higher eukaryotes. Budding yeast offers a simpler system in which to study the mechanism by which HU affects replication. Yeast possess no deoxyribonucleoside kinase activities, and thus deoxyribonucleotide synthesis is entirely dependent on ribonucleotide reductase. Also, yeast can easily be synchronized in G 1 using mating pheromone, and the availability of several temperature-sensitive cdc mutations allows cell cycle progression to be reversibly halted at specific points throughout the cell cycle (8). Reciprocal switch experiments in yeast have ordered the execution point of several cdc genes with respect to the HU-sensitive step during the cell cycle (9). Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the gene...

The transcription factors SBF and DSC1/MBF bind SCB and MCB promoter elements, respectively, and are essential for the cell cycle progression of Saccharomyces cerevisiae through the control of G1 cyclin gene expression. We isolated a gene (BRY1; Bacterial Response regulator in Yeast) able to activate either MCB or SCB promoter elements on a reporter plasmid which, when overexpressed, can bypass the normally essential requirement for SBF and DSC1/MBF by the stimulation of CLN1 and CLN2 expression. In the case of CLN2 at least, this expression depends upon the MCB and SCB promoter elements. In wild‐type yeast, the disruption of BRY1 has no apparent phenotype, but under conditions where the activities of SBF and DSC1/MBF are reduced, BRY1 becomes essential. Our data imply the existence of a third pathway affecting cyclin expression. BRY1 is the same gene as SKN7 which has significant sequence homology to the receiver domains found in response regulator proteins from the bacterial two‐component signal transduction pathways. SKN7 is thought to affect cell wall structure, and when highly overexpressed we find that BRY1/SKN7 is lethal perhaps because of perturbations in cell wall biosynthesis. The lethality is partially rescued by genes from the protein kinase C pathway, but genetic data imply that BRY1/SKN7 and protein kinase C are not in the same pathway. Our results suggest that Bry1/Skn7 can influence the expression of MCB‐ and SCB‐driven gene expression in budding yeast, perhaps including genes involved in cell wall metabolism, via a two‐component signal transduction pathway which activates Bry1/Skn7 in response to an unidentified signal.

BackgroundMetabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins.Principal FindingsHere we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each.ConclusionsChronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.

Thioredoxin reductases (Txnrd)1 maintain intracellular redox homeostasis in most organisms. Metazoans Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1 −/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd −/− and txnrd +/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells.