PPARgamma coactivator 1alpha (PGC-1alpha) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1alpha on the metabolism of ROS. PGC-1alpha is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1alpha is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1alpha null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1alpha levels dramatically protects neural cells in culture from oxidative-stressor-mediated death. These studies reveal that PGC-1alpha is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.

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Toxin-antitoxin (TA) modules are pairs of genes in which one member encodes a toxin that is neutralized or whose synthesis is prevented by the action of the product of the second gene, an antitoxin, which is either protein or RNA. We now report the identification of a TA module in the chromosome of Bacillus subtilis in which the antitoxin is an antisense RNA. The antitoxin, which is called RatA (for RNA antitoxin A), is a small (222 nucleotides), untranslated RNA that blocks the accumulation of the mRNA for a toxic peptide TxpA (for toxic peptide A; formerly YqdB). The txpA and ratA genes are in convergent orientation and overlap by ca. 75 nucleotides, such that the 3 region of ratA is complementary to the 3 region of txpA. Deletion of ratA led to increased levels of txpA mRNA and lysis of the cells. Overexpression of txpA also caused cell lysis and death, a phenotype that was prevented by simultaneous overexpression of ratA. We propose that the ratA transcript is an antisense RNA that anneals to the 3 end of the txpA mRNA, thereby triggering its degradation.

Induced expression of neuroprotective genes is essential for maintaining neuronal integrity after stressful insults to the brain. Here we show that NR4A nuclear orphan receptors are induced after excitotoxic and oxidative stress in neurons, up-regulate neuroprotective genes, and increase neuronal survival. Moreover, we show that NR4A proteins are induced by cAMP response element binding protein (CREB) in neurons exposed to stressful insults and that they function as mediators of CREB-induced neuronal survival. Animals with null mutations in three of six NR4A alleles show increased oxidative damage, blunted induction of neuroprotective genes, and increased vulnerability in the hippocampus after treatment with kainic acid. We also demonstrate that NR4A and the transcriptional coactivator PGC-1α independently regulate distinct CREB-dependent neuroprotective gene programs. These data identify NR4A nuclear orphan receptors as essential mediators of neuroprotection after exposure to neuropathological stress.excitotoxicity | kainic acid | oxidative stress N europathological conditions including stroke, Alzheimer's disease, and Parkinson's disease are associated with excitotoxic and oxidative stress. Transcriptional increases of neuroprotective genes, including antiapoptotic factors and scavengers of reactive oxygen species (ROS), are an important strategy for neuroprotection. Thus, understanding how neuroprotective gene programs are controlled at the transcriptional level is of considerable importance and may contribute to the identification of therapeutic strategies of disorders associated with neurodegeneration. cAMP response element binding protein (CREB) is a transcription factor that is activated in response to stressful stimuli such as hypoxia, oxidative stress, excitotoxicity, and ischemia (1). Evidence from loss-of-function and other types of experiments shows that CREB plays an important role in neuronal survival (2-5) and neuroprotection (6). It is also well established that CREB is required for acquisition of ischemic tolerance, an endogenous neuroprotective mechanism whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia (7,8).Despite the well-documented neuroprotective effect of CREB, only little is known of how CREB mediates this activity and only few directly regulated neuroprotective target genes have been identified (9-13). In addition to target genes that are directly neuroprotective, CREB-induced transcription factors or cofactors may also contribute to neuron survival by regulating downstream gene batteries controlled by elevated cAMP levels in a transcription factor cascade initiated by activated CREB. Indeed, CREB induces the expression of peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1α), an important regulator of ROS-detoxifying enzyme gene expression (14). However, how CREB mediates neuroprotective gene cascades via the induction of additional transcriptional regulators remains unexplored.The NR4A orphan nuclear receptor (NR...

The process of sporulation in the bacterium Bacillus subtilis is known to involve the programmed activation of several hundred protein-coding genes. Here we report the discovery of previously unrecognized genes under sporulation control that specify small, non-protein-coding RNAs (sRNAs). Genes for sRNAs were identified by transcriptional profiling with a microarray bearing probes for intergenic regions in the genome and by use of a comparative genomics algorithm that predicts regions of conserved RNA secondary structure. The gene for one such sRNA, SurA, which is located in the region between yndK and yndL, was induced at the start of development under the indirect control of the master regulator for entry into sporulation, Spo0A. The gene for a second sRNA, SurC, located in the region between dnaJ and dnaK, was switched on at a late stage of sporulation by the RNA polymerase sigma factor K , which directs gene transcription in the mother cell compartment of the developing sporangium. Finally, a third intergenic region, that between polC and ylxS, which specified several sRNAs, including two transcripts produced under the control of the forespore-specific sigma factor G and a third transcript generated by K , was identified. Our results indicate that the full repertoire of sporulation-specific gene expression involves the activation of multiple genes for small, noncoding RNAs.Protein-coding genes are the traditional focus of interest in microbial genetics, but increasing evidence points to the importance of genes that specify small, untranslated RNAs (sRNAs) (40). sRNAs range in size from approximately 30 nucleotides (nt) to 600 nt in bacteria, and in a growing number of cases functions have been elucidated for specific sRNAs. For example, in Escherichia coli, where sRNAs have been studied most extensively, functions have been reported for 15 of the approximately 80 sRNAs so far identified (12,19). Among these are 6S RNA, which binds to and inhibits RNA polymerase containing 70 and was found to play a role in long-term cell survival (42); OxyS, which is induced under oxidative stress and regulates the expression of at least 40 genes, including the mRNAs for the transcriptional regulator FhlA and the stationary-phase sigma factor S (1, 2); DsrA, which enhances the translation of the mRNA for S and inhibits the translation of the mRNA for the global repressor H-NS (22, 38); and finally, RyhB, which targets mRNAs for proteins involved in the tricarboxylic acid cycle and iron storage and the mRNA for superoxide dismutase (28,29). sRNAs have been detected in other bacteria, such as Staphylococcus aureus (18), Pseudomonas aeruginosa (45), Vibrio cholerae (7, 24), and Borrelia burgdorferi (32).sRNAs have also been reported for the spore-forming bacterium Bacillus subtilis, the subject of this report. Examples of sRNA that are similar to small, untranslated RNAs found in non-spore-forming genera are SsrA (21), small cytoplasmic RNA (31), P RNA (26), two 6S-like RNAs called 6S-1 and 6S-2 (3, 5, 41-43), and a 5S-like RN...

Purpose: Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARg ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARg/carboplatin combination in these more relevant models of drug resistant non^small cell lung cancer. Experimental Design: Tumorigenesis was induced by activation of either mutant KRAS or EGFR. Mice then received either rosiglitazone or carboplatin monotherapy, or a combination of both drugs. Change in tumor burden, pathology, and evidence of apoptosis and cell growth were assessed. Results: Tumor burden remained unchanged or increased in the mice after monotherapy with either rosiglitazone or carboplatin. In striking contrast, we observed significant tumor shrinkage in mice treated with these drugs in combination. Immunohistochemical analyses showed that this synergy was mediated via both increased apoptosis and decreased proliferation. Importantly, this synergy between carboplatin and rosiglitazone did not increase systemic toxicity. Conclusions: These data show that the PPARg ligand/carboplatin combination is a new therapy worthy of clinical investigation in lung cancers, including those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy.Lung cancer is the leading cause of cancer-related deaths. There are over 210,000 cases of lung cancer diagnosed and over 160,000 deaths in the United States alone (1, 2). The most common type of lung cancer is non -small cell lung cancer (NSCLC), which comprises over 75% of the cases (3). Despite advances in multimodality therapies, <15% of patients with NSCLC survive beyond 5 years of initial diagnosis. Activating mutations of the KRAS proto-oncogene are among the most common genetic alterations in NSCLC (4 -8). These mutations lead to the constitutive activation of downstream signaling transduction pathways including RAF and phosphatidylinositol-3-OH kinase. These pathways, in turn, regulate proliferation and survival. In addition to playing a role in the development of lung cancer, mutations in KRAS predict a poor outcome and a poor response to conventional therapy such as platinumbased drugs, as well as targeted therapy (4, 9 -12). The

Multiple mechanisms likely contribute to neuronal death in Parkinson’s disease (PD), including mitochondrial dysfunction and oxidative stress. Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1α) positively regulates the expression of genes required for mitochondrial biogenesis and the cell’s antioxidant responses. Also, expression of PGC-1α-regulated genes is low in substantia nigra (SN) neurons in early PD. Thus upregulation of PGC-1α is a candidate neuroprotective strategy in PD. Here, an adeno-associated virus (AAV) was used to induce unilateral overexpression of Pgc-1α, or a control gene, in the SN of wild-type C57BL/6CR mice. Three weeks after AAV administration, mice were treated with saline or MPTP. Overexpression of Pgc-1α in the SN induced expression of target genes, but unexpectedly it also greatly reduced the expression of tyrosine hydroxylase (Th) and other markers of the dopaminergic phenotype with resultant severe loss of striatal dopamine. Reduced Th expression was associated with loss of Pitx3, a transcription factor that is critical for the development and maintenance of dopaminergic cells. Expression of the neurotrophic factor Bdnf, which also is regulated by Pitx3, similarly was reduced. Overexpression of Pgc-1α also led to increased sensitivity to MPTP-induced death of Th+ neurons. Pgc-1α overexpression alone, in the absence of MPTP treatment, did not lead to cell loss in the SN or to loss of dopaminergic terminals. These data demonstrate that overexpression of Pgc-1α results in dopamine depletion associated with lower levels of Pitx3 and enhances susceptibility to MPTP. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in PD.

The Bacillus subtilis transcription factor E directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven E -controlled genes cause a mild (2-to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN. Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.