Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside.
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The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the effects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to sufficient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK. (+info)
5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved.
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5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells. (+info)
A new synthesis of inosine from 5-amino-1-beta-D-ribofuranosyl-4-imidazole-carboxamide.
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Inosine was prepared (15% yield) by treatment of 5-amino-1-beta-D-ribofuranosyl-4-imidazolecarboxamide (AICA-riboside) with chloroform in the presence of sodium methoxide. This ring closure can be reasonably explained by assuming the formation of dichlorocarbene from chloroform and alkali. Carbon tetrachloride or hexachloroethane as a carbene source was more effective for the ring closure of AICA-riboside, giving inosine in 48% and 51% yields respectively. (+info)
Synthesis of guanosine and its derivatives from 5-amino-1-beta-D-ribofuranosyl-4-imidazolecarboxamide. III. Formation of a novel cycloimidazole nucleoside and its cleavage reactions.
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A new cycloimidazole nucleoside, 5-(1 inch -benzamido-1 inch-hydroxymethylene) amino-2', 1 inch-anhydro-1-beta-D-ribofuranosyl-4-imidazolecarboxamide (III) was synthesized by reaction of 5-amino-1-beta-D-ribofuranosyl-4-imidazolecarboxamide (AICA-riboside) with benzoyl isothiocyanate followed by methylation with methyl iodide. The structure of III was elucidated on the basis of its nmr spectra and chemical reactions. Of special interest are reactions of III with various nucleophiles. For example, guanosine (IX) was obtained by amination of III wtih ammonia in 72% yield. Analogous reactions of III with methylamine and dimethylamine gave N2-methylguanosine (X) and N2-dimethylguanosine (XI), respectively. Refluxing of III in alkaline solution afforded xanthosine (VII). The probable mechanism of formation and facile ring-opening of III is also discussed. (+info)
Synthesis of guanosine and its derivatives from 5-amino-1-beta-D-ribofuranosyl-e-imidazolecarboxamide. IV. A new route to guanosine via cyanamide derivative.
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4-Cyanamido-5-imidazolecarboxamide (IV) was prepared by brief treatment of 5-(S-methylisothiocarbamoyl) amino-4-imidazolecarboxamide (V) with alkali. Compound VI was converted in an alkaline solution to either guanine (VII) or isoguanine (VIII), depending on the concentration of alkali. This procedure was applied to the synthesis of 2',3'-0-isopropylideneguanosine (XVI) from the riboside of 5-(N'-benzoyl-S-methylthiocarbamoyl) amino-4-imidazolecarboxamide (IX), PROviding a new route to XVI. (+info)
An improved method for the separation and quantitation of the modified nucleosides of transfer RNA.
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A method is described which allows a very efficient determination of the modified nucleosides of tRNA. The technique involves enzymatic degradation of the tRNA to nucleosides at pH 7.6 and their separation by two-dimensional thin-layer chromatography on cellulose-coated aluminum foils. Based on the analysis of two mammalian tRNAs it is shown that the technique is suitable for the determination of chemically unstable nucleosides as well as the ribose-methylated compounds. At least 36 of the 45 known modified nucleosides can be separated and quantitatively determined by the method described. This procedure is especially suitable for the estimation of the nucleoside composition of unlabeled tRNAs as well as for studying the post-transcriptional modifications of tRNA. (+info)
Acute vascular and interstitial rejection following renal allograft transplantation in dogs.
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Renal allograft transplantation was performed in four beagles. Immunosuppressive treatment using cyclosporine, mizoribin and prednisolone was continued from Day 5 pre- until Day 20 post-transplantation. Between Days 28 and 32 post-transplantation, an abrupt elevation of the serum creatinine values followed by the development of uremia was seen in all recipients. Histopathology of the allografts examined between Days 28 and 37 revealed edema, necrosis, hemorrhage and severe diffuse interstitial cellular infiltration as well as tubulitis. Glomerular changes notably included swelling of the tufts due to hypercellularity, which was consistent with transplant glomerulitis. The intrarenal arteries exhibited fibrinoid necrosis of the walls and intimal or transmural cellular infiltration. These renal lesions were consistent with those of acute vascular and interstitial rejection in humans. (+info)
Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes.
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Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment. (+info)