Synthesis of α-Adenosine 5′-Monophosphate

The distributions of protein, calcium and inorganic phosphate among casein micelles of skimmilk before and after frozen storage were investigated by gel filtration through Sephadex G-200 with 6.6 m urea solution as eluant.

The results showed that the native casein micelles were fractionated into two fractions. Fast eluting fraction contained large amount of calcium and inorganic phosphorus. Slow eluting fraction contained calcium but was essentially free of inorganic phosphorus. Frozen storage caused the increase of proportion of the fast eluting fraction accompanying the increase of calcium and inorganic phosphorus contents in it. It is suggested that the salt linkage newly formed during frozen storage causes the increase of proportion of the fast eluting fraction.     

RIG-I Selectively Discriminates against 5′-Monophosphate RNA

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Synthesis and Biological Activity of BIS(Pivaloyloxymethyl) Ester of 2′-Azido-2′-Deoxyuridine 5′-Monophosphate

Abstract

Bis(pivaloyloxymethyl) ester of 2′-azido-2′-deoxyuridine 5′-monophosphate was prepared as a prodrug to generate 2′-azido-2′-deoxyuridine 5′-diphosphate inside the cell. A synthetic route utilizing stannyl phosphate was adopted in the preparation. The prodrug was evaluated for cell growth inhibition against a variety of tumor cell lines along with 2′-azido-2′-deoxyuridine and 2′-azido-2′-deoxycytidine.     

Concentration of Cyclic Adenosine 3′,5′ -Monophosphate in Human Leucocytes during Phagocytosis

CYCLIC adenosine 3′,5′-monophosphate (cyclic AMP) has been established as a mediator of various hormonal effects in the appropriate target cells¹. Adenyl cyclase converts adenosine triphosphate (ATP) to cyclic AMP and is widely distributed in the membrane of mammalian nucleated cells^2–4. Since the early process of phagocytosis involves the physical and chemical contact of the cell membrane to the objects and subsequent formation of phagosome, we postulated that one of the earliest biochemical changes during phagocytosis might be an activation of adenyl cyclase and an alteration of concentrations of cyclic AMP in the phagocytes.     

Overexpression of Inosine 5′-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells

Background

Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5′-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance.

Methodology/Principal Findings

Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2.

Conclusions

IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.     

Characterization of a Bifunctional Cytidine 5′-Monophosphate N-acetylneuraminic acid Synthetase Cloned from Streptococcus Agalactiae

Recombinant CMP-sialic acid synthetase, cloned from Streptococcus agalactiae serotype V strain 2603 V/R, is bifunctional having both CMP-sialic acid synthetase and acetylhydrolase (acylesterase) activities. The enzyme is active over a wide pH range with an optimal CMP-sialic acid synthetase activity at pH 9.0 and an optimal acetylhydrolase activity at pH 8.0. A metal cofactor (either Mg²⁺ or Mn²⁺) is required for the CMP-sialic acid synthetase activity but is not for acetylhydrolase activity. Both catalytic functions, however, are impaired by high concentrations of Mn²⁺. Received 10 August 2005; Revisions requested 30 August 2005; Revisions received 1 November 2005; Accepted 2 November 2005     

Synthesis and Activity of Modified Cytidine 5′-Monophosphate Probes for T4 RNA Ligase 1

We describe the synthesis of a series of unique base modified ligation probes such as p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 and tested their biological activity with T4 RNA ligase 1 using a standard pCp probe 1 as a control. The intermolecular ligation assay was developed using a 5′-FAM labeled 24 mer single-stranded (ss) RNA and the average ligation efficiencies for pCp 1, p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 were found to be 44%, 81%, 39% and 16% respectively, as determined using a denaturing gel analysis. Furthermore, confirmation of the ligation activity of the biotinylated probes to the RNA substrate was confirmed by streptavidin conjugation and analysis by nondenaturing gel electrophoresis. These results strongly suggest that the new probes are valid substrates for T4 RNA ligase 1 and therefore could be useful for developing a miRNA detection system that includes rapid isolation, efficient labeling and detection of miRNAs on sensitivity-enhanced microarrays.     

Modulation of Adenosine 5′-Monophosphate Adsorption Onto Aqueous Resident Pyrite: Potential Mechanisms for Prebiotic Reactions

The adsorption of adenosine 5-monophosphate (5-AMP) ontopyrite (FeS₂) and its modulation by acetate, an organic precursor of complex metabolic pathways, was studied in aqueousmedia that simulate primitive environments. 5-AMP adsorptionrequires divalent cations, indicating that a cationic bridge mediates its attachment to negatively charged sites of the mineral surface. The isotherm of 5-AMP adsorption exhibits a strong cooperative effect at low nucleotide concentrations inacetate-rich medium, whereas high levels of adsorption were only found at high nucleotide concentrations in a model of primitive seawater (acetate free). The modulating role of acetate is also evidenced in the presence of high dipolar moment molecules: dimethyl sulfoxide (Me₂SO) and dimethylformamide (DMF) strongly inhibit 5-AMP adsorption in acetate-rich media, whereas no effect of DMF was found in artificial seawater. The observation that exogenous divalentcations are not needed for acetate attachment onto FeS₂ reveals that organic acids can interact with the Fe²⁺ atoms in the mineral surface. All considered, the results showthat complex and flexible iron-sulfide/biomonomers interactionscan be modulated by molecules that accumulate in the interfacelayer.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Disordered Single Crystal Evidence for a Quadruple Helix Formed by Guanosine 5′-Monophosphate

Abstract

The disodium salt of guanosine 5′-monophosphate (5′-GMP) has been crystallized earlier in an orthorhombic array. We have obtained a new crystal form of 5′-GMP at pH 8 which reveals a clear helical nature, with guanine bases stacked perpendicular to the helix axis. Although the X-ray pictures show partial disorder, they can be indexed on a hexagonal net with a = b = 28.6 Å,c = 9.8 Å, V= 6942ų(1Å = 0.1 nm). The probable space group is P6₄, and past experience with ca. 600 ų per base in oligonucleotide crystals suggests that the cell contains 12 GMP molecules. The crystal packing parameters and the intensity distribution agree with a model of three hydrogen-bonded guanine tetrads in the unit cell, stacked so as to build a quadruple helix similar to that proposed earlier from fiber studies (Zimmerman, S.B., J. Mol. Biol. 106, 663–672 (1976)).     

Synthesis of 5′-Deoxy-5′-nucleosideacetic Acid Derivatives

Abstract

The synthesis of several new 5′-deoxy-5′-nucleosideacetic acid derivatives by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5′-aldehydes is described.     

5′-Halogenated Formycins as Inhibitors of 5′-Deoxy-5′-methylthioadenosine Phosphorylase: Protection of Cells Against the Growth-Inhibitory Activity of 5′-Halogenated Adenosines

Abstract

A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with K_i values in the range of 10^?7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.     

Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.     

Synthesis of 5′-Fluoro-5′-deoxy-and 5′-Amino-5′-Deoxytoyocamycin and Sangivamycin and Some Related Derivatives

Abstract

A series of 5′-substituted analogs of toyocamycin were prepared by condensation of silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with protected 5-azido-5-deoxy- or 5-fluoro-5-deoxyribofuranose followed by debromination and deblocking. Alternatively, 5′-azido-5′-deoxytoyocamycin was prepared by azidation of toyocamycin. Conversion of the 5-nitrile function of the toyocamycin derivatives into a carboxamide or a thiocarboxamide gave the corresponding analogs of sangivamycin or thiosangivamycin while reduction of the 5′-azido-5′-deoxy nucleosides provided 5′-amino-5′-deoxy derivatives.     

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

Template-directed oligomerization of 5′-deoxy-5′-nucleosideacetic acid derivatives

5-Deoxy-5-nucleosideacetic acids II–V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.     

Inhibition of Adenovirus Type 12 Induced DNA Synthesis in G1-arrested BHK21 Cells by Dibutyryl Adenosine Cyclic 3′ : 5′-Monophosphate

THE intracellular concentration of adenosine cyclic 3′ : 5′-monophosphate (cAMP) has been shown to increase in cells reaching confluency, that is, under conditions of contact inhibition and it has been proposed that contact inhibition may be mediated by the activation of adenyl cyclase¹. Because loss of contact inhibition is one of the important characteristics of transformed cells, it is possible that variations in the level of cAMP play a crucial role in events associated with cell transformation. Growth of tumorigenic cell lines has been shown to be inhibited (80–90%) in the presence of cAMP¹ and adenyl cyclase activity has been found to be much reduced in polyoma transformed cells². BHK21 cells have been shown to be arrested in the G1-phase of the cell cycle after growth for 48 h in medium supplemented with serum at a low concentration (0.5%)³. BHK21 cells are non-permissive for infection with oncogenic adenovirus type 12 (Ad 12). Infection of a G1-arrested cell population with Ad12 results in induction of cellular DNA replication⁴, early virus mRNA is transcribed⁵, infected cells synthesize T antigen⁶, but no viral DNA synthesis⁷ late mRNA⁸, or viral capsid proteins⁸ can be detected after infection. Infection induces the cells to enter mitosis but the chromosomes break down and most of the cells die⁶. As a result, DNA synthesis is limited to a single burst^4,6. Here we report the effects of dibutyryl-cAMP on induction of cellular DNA synthesis in G1-arrested BHK21 cells by infection with Adl2 or serum stimulation.     

Synthesis of 3′-Amino-3′-deoxyguanosine 5′-Triphosphate

Abstract

Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N²-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH₂).     

Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.