Regulation of multimers via truncated isoforms: a novel mechanism to control nitric-oxide signaling

Nitric oxide (NO) is an essential regulator of Drosophila development and physiology. We describe a novel mode of regulation of NO synthase (NOS) function that uses endogenously produced truncated protein isoforms of Drosophila NOS (DNOS). These isoforms inhibit NOS enzymatic activity in vitro and in vivo, reflecting their ability to form complexes with the full-length DNOS protein (DNOS1). Truncated isoforms suppress the antiproliferative action of DNOS1 in the eye imaginal disc by impacting the retinoblastoma-dependent pathway, yielding hyperproliferative phenotypes in pupae and adult flies. Our results indicate that endogenous products of the dNOS locus act as dominant negative regulators of NOS activity during Drosophila development

noxin, a novel stress-induced gene involved in cell cycle and apoptosis

We describe a novel stress-induced gene, noxin, and a knockout mouse line with an inactivated noxin gene. The noxin gene does not have sequelogs in the genome and encodes a highly serine-rich protein with predicted phosphorylation sites for ATM, Akt, and DNA-dependent protein kinase kinases; nuclear localization signals; and a Zn finger domain. noxin mRNA and protein levels are under tight control by the cell cycle. noxin, identified as a nitric oxide-inducible gene, is strongly induced by a wide range of stress signals: gamma- and UV irradiation, hydrogen peroxide, adriamycin, and cytokines. This induction is dependent on p53. Noxin accumulates in the nucleus in response to stress and, when ectopically expressed, Noxin arrests the cell cycle at G1; although it also induces p53, the cell cycle arrest function of Noxin is independent of p53 activity. noxin knockout mice are viable and fertile; however, they have an enlarged heart, several altered hematopoietic parameters, and a decreased number of spermatids. Importantly, loss or downregulation of Noxin leads to increased cell death. Our results suggest that Noxin may be a component of the cell defense system: it is activated by various stress stimuli, helps cells to withdraw from cycling, and opposes apoptosis

Nitric oxide negatively regulates mammalian adult neurogenesis

Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system

Essential function of nitric oxide synthase in Drosophila

Nitric oxide (NO), produced by NO synthases (NOS), is a short-lived intra- and transcellular messenger that regulates many physiological functions in vertebrates and invertebrates (e.g., blood pressure, muscle contraction, innate immunity, cell division and differentiation, response to hypoxia, and memory formation [1]). Given its numerous functions, it might be expected that a lack of NOS activity would be lethal for the developing organism. However, such an indispensable role for NO in development has not yet been demonstrated. A genetic analysis of NO function in vertebrates is complicated by the presence of three NOS genes. Mice with a homozygous ablation of any single NOS gene are viable, animals with two NOS genes knocked out show drastically reduced viability and triple knockout animals have not yet been generated 2., 3., 4., 5. and 6.

The Drosophila nitric-oxide synthase gene (dNOS) encodes a family of proteins that can modulate NOS activity by acting as dominant negative regulators

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. We cloned and analyzed the only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS). It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. We identified eight new transcripts that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5'-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene lead to premature termination of the open reading frame. As a result, none of the alternative transcripts encode an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, we demonstrate that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. Our results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene

Nitric oxide interacts with the retinoblastoma pathway to control eye development in Drosophila

Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development [1,2]. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway [3,4], which integrates signals from multiple effecters ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger [5], can reversibly suppress DNA synthesis and cell division [6,7]. In developing flies, the antiproliferative action of NO is. essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly [8-10]. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. We used transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors to address this issue. We found that manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. Our data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway

Axonal trafficking of an antisense RNA transcribed from a pseudogene is regulated by classical conditioning

Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory. We show the antiNOS-2 RNA is axonally trafficked and demonstrate that this is regulated by classical conditioning. Critically, a single conditioning trial changes the amount of antiNOS-2 RNA transported along the axon. This occurs within the critical time window when neurotransmitter NO is required for memory formation. Our data suggest a role for the antiNOS-2 RNA in establishing memories through the regulation of NO signaling at the synapse

Patiromer in patients with kidney disease and hyperkalemia receiving RAAS inhibitors

BACKGROUND: Hyperkalemia increases the risk of death and limits the use of inhibitors of the renin-angiotensin-aldosterone system (RAAS) in high-risk patients. We assessed the safety and efficacy of patiromer, a nonabsorbed potassium binder, in a multicenter, prospective trial. METHODS: Patients with chronic kidney disease who were receiving RAAS inhibitors and who had serum potassium levels of 5.1 to less than 6.5 mmol per liter received patiromer (at an initial dose of 4.2 g or 8.4 g twice a day) for 4 weeks (initial treatment phase); the primary efficacy end point was the mean change in the serum potassium level from baseline to week 4. Eligible patients at the end of week 4 (those with a baseline potassium level of 5.5 to <6.5 mmol per liter in whom the level decreased to 3.8 to <5.1 mmol per liter) entered an 8-week randomized withdrawal phase in which they were randomly assigned to continue patiromer or switch to placebo; the primary efficacy end point was the between-group difference in the median change in the serum potassium level over the first 4 weeks of that phase. RESULTS: In the initial treatment phase, among 237 patients receiving patiromer who had at least one potassium measurement at a scheduled visit after day 3, the mean (±SE) change in the serum potassium level was -1.01±0.03 mmol per liter (P<0.001). At week 4, 76% (95% confidence interval, 70 to 81) of the patients had reached the target potassium level (3.8 to <5.1 mmol per liter). Subsequently, 107 patients were randomly assigned to patiromer (55 patients) or placebo (52 patients) for the randomized withdrawal phase. The median increase in the potassium level from baseline of that phase was greater with placebo than with patiromer (P<0.001); a recurrence of hyperkalemia (potassium level, ≥5.5 mmol per liter) occurred in 60% of the patients in the placebo group as compared with 15% in the patiromer group through week 8 (P<0.001). Mild-to-moderate constipation was the most common adverse event (in 11% of the patients); hypokalemia occurred in 3%. CONCLUSIONS: In patients with chronic kidney disease who were receiving RAAS inhibitors and who had hyperkalemia, patiromer treatment was associated with a decrease in serum potassium levels and, as compared with placebo, a reduction in the recurrence of hyperkalemi