Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies.

A common thread among conserved life span regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of AMP biosynthetic enzymes extend Drosophila life span. The life span benefit of these mutations depends upon increased AMP:ATP and ADP:ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended life span, while AMPK RNAi reduced life span. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed life span extension. Remarkably, this simple change in diet also blocked the prolongevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult life span; provide a mechanistic link between cellular anabolism and energy sensing pathways; and indicate that dietary adenine manipulations might alter metabolism to influence animal life span.

Interleukin-22 modulates gut epithelial and immune barrier functions following acute alcohol exposure and burn injury.

Interleukin-22 (IL-22) maintains gut epithelial integrity and expression of antimicrobial peptides Reg3ß and Reg3γ. Our laboratory has shown that acute alcohol/ethanol (EtOH) exposure before burn injury results in increased gut permeability, intestinal T-cell suppression, and enhanced bacterial translocation. Herein, we determined the effect of combined EtOH intoxication and burn injury on intestinal levels of IL-22 as well as Reg3ß and Reg3γ expression. We further examined whether in vivo restitution of IL-22 restores gut permeability, Reg3ß and Reg3γ levels, and bacterial load (e.g., gut bacterial growth) within the intestine after EtOH and burn injury. Male mice, ∼25g, were gavaged with EtOH (2.9 mg/kg) before receiving a ∼12.5% total-body-surface-area, full-thickness burn. Mice were immediately treated with saline control or IL-22 (1 mg/kg) by i.p. injection. One day after injury, there was a significant decrease in intestinal IL-22, Reg3ß, and Reg3γ expression along with an increase in intestinal permeability and gut bacterial load after EtOH combined with burn injury, as compared with sham injury. Treatment with IL-22 normalized Reg3ß and Reg3γ expression and attenuated the increase in intestinal permeability after EtOH and burn injury. Qualitatively, IL-22 treatment reduced the bacterial load in nearly half of mice receiving EtOH combined with burn injury. Our data indicate that IL-22 maintains gut epithelial and immune barrier integrity after EtOH and burn injury; thus, the IL-22/antimicrobial peptide pathway may provide a therapeutic target for the treatment of patients who sustain burn injury under the influence of EtOH.

Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices.

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5'-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-(14)C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.

Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae.

Ty1 retrotransposons of the yeast Saccharomyces cerevisiae are activated by different kinds of stress. Here we show that Ty1 transcription is stimulated under severe adenine starvation conditions. The Bas1 transcriptional activator, responsible for the induction of genes of the de novo AMP biosynthesis pathway (ADE) in the absence of adenine, is not involved in this response. Activation occurs mainly on Ty1 elements, whose expression is normally repressed by chromatin and is suppressed in a hta1-htb1Delta mutant that alters chromatin structure. Activation is also abolished in a snf2Delta mutant. Several regions of the Ty1 promoter are necessary to achieve full activation, suggesting that full integrity of the promoter sequences might be important for activation. Together, these observations are consistent with a model in which the activation mechanism involves chromatin remodeling at Ty1 promoters. The consequence of Ty1 transcriptional activation in response to adenine starvation is an increase in Ty1 cDNA levels and a relief of Ty1 dormancy. The retrotransposition of four native Ty1 elements increases in proportion to their increase in transcription. Implications for the regulation of Ty1 mobility by changes in Ty1 mRNA levels are discussed.

4-Aminoquinolines: novel nociceptin antagonists with analgesic activity.

Small-molecule nociceptin antagonists were synthesized to examine their therapeutic potential. After a 4-aminoquinoline derivative was found to bind with the human ORL(1) receptor, a series of 4-aminoquinolines and related compounds were synthesized and their binding was evaluated. Elucidation of structure-activity relationships eventually led to the optimum compounds. One of these compounds, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (11) not only antagonized nociceptin-induced allodynia in mice but also showed analgesic effect in a hot plate test using mice and in a formalin test using rats. Its analgesic effect was not antagonized by the opioid antagonist naloxone. These results indicate that this nociceptin antagonist has the potential to become a novel type of analgesic that differs from mu-opioid agonists.

Adenylosuccinate synthetase: site of action of hydantocidin, a microbial phytotoxin.

The site of action of hydantocidin was probed using Arabidopsis thaliana plants growing on agar plates. Herbicidal effects were reversed when the agar medium was supplemented with AMP, but not IMP or GMP, suggesting that hydantocidin blocked the two-step conversion of IMP to AMP in the de novo purine biosynthesis pathway. Hydantocidin itself did not inhibit adenylosuccinate synthetase or adenylosuccinate lyase isolated from Zea mays. However, a phosphorylated derivative of hydantocidin, N-acetyl-5'-phosphohydantocidin, was a potent inhibitor of the synthetase but not of the lyase. These results identify the site of action of hydantocidin and establish adenylosuccinate synthetase as an herbicide target of commercial potential.

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe.

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.