Extracellular Uridine Nucleotides-Induced Contractions Were Increased in Femoral Arteries of Spontaneously Hypertensive Rats

<b><i>Introduction:</i></b> Femoral arterial dysfunction including abnormal vascular responsiveness to endogenous ligands was often seen in arterial hypertension. Extracellular nucleotides including uridine 5′-diphosphate (UDP) and uridine 5′-triphosphate (UTP) play important roles for homeostasis in the vascular system including controlling the vascular tone. However, responsiveness to UDP and UTP in femoral arteries under arterial hypertension remains unclear. The aim of this study was to investigate if hypertension has an effect of vasoconstrictive responsiveness to UDP and UTP in femoral arteries of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) after 7 and 12 months old. <b><i>Methods:</i></b> Organ baths were conducted to determine vascular reactivity in isolated femoral arterial rings. <b><i>Results:</i></b> In femoral arteries obtained from 12-month-old rats, augmented contractile responses to UDP and UTP were seen in femoral arteries of SHR than in those of WKY under situations not only intact but also nitric oxide synthase inhibition, whereas no difference of extracellular potassium-induced vasocontraction was seen in both SHR and WKY groups. Similar contraction trends occurred in femoral arteries obtained from 7-month-old rats. Moreover, contractions induced by UDP and UTP were increased in endothelium-denuded arteries. Cyclooxygenase inhibition decreased the contractions induced by these nucleotides and abolished the differences in responses between the SHR and WKY groups. <b><i>Conclusions:</i></b> This study demonstrates the importance of regulation of extracellular uridine nucleotides-induced contractions in hypertension-associated peripheral arterial diseases.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates

Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data—in combination with in vivo results—suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification. Key points •PPK1ATand PPK2AThave different substrate specificities, •PPK2ATis a subgroup 1 member of PPK2s, •PPK2ATcatalyzes the formation of polyphosphorylated nucleosides

Thrombin binding aptamer G-quadruplex stabilized by pyrene-modified nucleotides

Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.

Polyuridylation in Eukaryotes: A 3′-End Modification Regulating RNA Life

In eukaryotes, mRNA polyadenylation is a well-known modification that is essential for many aspects of the protein-coding RNAs life cycle. However, modification of the 3′ terminal nucleotide within various RNA molecules is a general and conserved process that broadly modulates RNA function in all kingdoms of life. Numerous types of modifications have been characterized, which are generally specific for a given type of RNA such as the CCA addition found in tRNAs. In recent years, the addition of nontemplated uridine nucleotides or uridylation has been shown to occur in various types of RNA molecules and in various cellular compartments with significantly different outcomes. Indeed, uridylation is able to alter RNA half-life both in positive and in negative ways, highlighting the importance of the enzymes in charge of performing this modification. The present review aims at summarizing the current knowledge on the various processes leading to RNA 3′-end uridylation and on their potential impacts in various diseases.

APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition

Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition.

Endonuclease Associations with Three Distinct Editosomes in Trypanosoma brucei

Three distinct editosomes, typified by mutually exclusive KREN1, KREN2, or KREN3 endonucleases, are essential for mitochondrial RNA editing in Trypanosoma brucei. The three editosomes differ in substrate endoribonucleolytic cleavage specificity, which may reflect the vast number of editing sites that need insertion or deletion of uridine nucleotides (Us). Each editosome requires the single RNase III domain in each endonuclease for catalysis. Studies reported here show that the editing endonucleases do not form homodimeric domains, and may therefore function as intermolecular heterodimers, perhaps with KREPB4 and/or KREPB5. Editosomes isolated via TAP tag fused to KREPB6, KREPB7, or KREPB8 have a common set of 12 proteins. In addition, KREN3 is only found in KREPB6 editosomes, KREN2 is only found in KREPB7 editosomes, and KREN1 is only found in KREPB8 editosomes. These are the same associations previously found in editosomes isolated via the TAP-tagged endonucleases KREN1, KREN2, or KREN3. Furthermore, TAP-tagged KREPB6, KREPB7, and KREPB8 complexes isolated from cells in which expression of their respective endonuclease were knocked down were disrupted and lacked the heterotrimeric insertion subcomplex (KRET2, KREPA1, and KREL2). These results and published data suggest that KREPB6, KREPB7, and KREPB8 associate with the deletion subcomplex, whereas the KREN1, KREN2, and KREN3 endonucleases associate with the insertion subcomplex.

Vistonuridine: An orally administered, life-saving antidote for 5-fluorouracil (5FU) overdose

9616 Background: 5FU is widely used to treat solid tumors and is often administered via infusion pump at or near its maximum tolerated dose (MTD). Toxicities and even death can occur in patients over-exposed to 5FU. Uridine is a direct biochemical antagonist of 5FU toxicity; uridine nucleotides dilute intracellular fluorouridine nucleotides derived from 5FU, reducing their lethal incorporation into RNA. However, uridine is poorly bioavailable (7%) and is therefore not a clinically viable antidote. Vistonuridine is an orally administered prodrug of uridine that delivers approximately 8-fold more uridine than administration of uridine itself. Methods: 17 patients overdosed with 5FU have been treated with vistonuridine as an antidote. Patients received vistonuridine (10g q6h for 20 doses) beginning 8 to 96 hours after overdose. Data from 13 patients with similar 5FU overdoses provide the time course and outcomes for patients receiving available supportive care without vistonuridine. A severity score, integrating dose and infusion rate, was calculated for all the patients, and this tool could be used by healthcare workers to determine the expected severity and outcome of a 5FU overdose. Results: All 17 overdose patients treated with vistonuridine recovered fully. Most, especially those for whom vistonuridine treatment was initiated sooner, had relatively modest toxicity. In marked contrast, all 11 of the literature-reported cases of 5FU overdose for which an outcome of death would have been predicted died from the overdose despite receiving available supportive care. Conclusions: These clinical data illustrate the serious, debilitating and life threatening nature of 5FU overdose and the current lack of effective treatments. Vistonuridine appears to be a safe and effective, life-saving antidote to 5FU overdose. [Table: see text]