Assay of 2',5'-oligoadenylate phosphodiesterase activity in mouse L-cell extracts

A rapid and convenient assay for adenylyl(2' leads to 5')adenosine(A2'p5'A) or adenylyl(3' leads to 5')adenosine(A3'p5'A) phosphodiesterase activities is described. The dinucleotides A3'p5'A and A2'p5'A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3'p5'A or [3H]A2'p5'A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2'p5'A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2'p5'A as extracts from interferon pretreated cells.     

Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.     

Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family

2′,5′-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Abbreviations  The nomenclature of particular OASs in the present paper is based on the level of their sequence similarities. OAS1 refers to the OAS consisting of a single OAS domain. Capital letters (OAS1X) are used to differentiate between OAS1 types with sequence homologies of less than 50%; their variants are additionally marked in small letters (OASXx, sequence homology ~70 to ~95%). Labels prime and double prime denote sponge OAS1Xx gene haplotypes (sequence homology close to 100%, a few amino acid substitutions).     

A scientific journey through the 2-5A/RNase L system

The antiviral and antitumor actions of interferons are caused, in part, by a remarkable regulated RNA cleavage pathway known as the 2-5A/RNase L system. 2′-5′ linked oligoadenylates (2-5A) are produced from ATP by interferon-inducible synthetases. 2-5A activates pre-existing RNase L, resulting in the cleavage of RNAs within single-stranded regions. Activation of RNase L by 2-5A leads to an antiviral response, although precisely how this happens is a subject of ongoing investigations. Recently, RNase L was identified as the hereditary prostate cancer 1 gene. That finding has led to the discovery of a novel human retrovirus, XMRV. My scientific journey through the 2-5A system recounts some of the highlights of these efforts. Knowledge gained from studies on the 2-5A system could have an impact on development of therapies for important viral pathogens and cancer.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

The Influence of Gender and Age on Neonatal Rat Hypothalamic 5-HT1A and 5-HT2A Receptors

1.Rat hypothalamic 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) concentrations are transiently sexually differentiated in the second week postpartum (pp), with higher levels in the female. In this report we investigate the possibility that 5-HT receptors may also exhibit sexual dimorphism in the neonatal period.2.5-HT1A and 5-HT2A receptors were quantitated by radioligand binding of [³H]ketanserin and [³H]8-OH DPAT, respectively, in hypothalamus and amygdala from male and female rats at days 8–16 pp.3.There was no sexual dimorphism or change in the density of 5-HT2A binding in hypothalamus or amygdala over days 8–16 pp. There was also no sexual dimorphism of 5-HT1A receptors.4.There was an increase in 5-HT1A receptor density in both the hypothalamus and the amygdala. In the hypothalamus, but not the amygdala, this increase was interrupted on day 14 by a decrease in 5-HT1A receptors, which we suggest may be of physiological significance in modifying the eventual pattern of adult agonistic activity.5.The results suggest that the sexual dimorphism in 5-HT turnover is predominantly presynaptic, relating to altered synthesis and/or release, and is not of sufficient magnitude or duration to produce adaptive responses in postsynaptic 5-HT1A or 5-HT2A receptors.     

The specificity of monoclonal antibody A2B5 to c-series gangliosides

To examine the specificity of monoclonal antibody A2B5, four A2B5-reactive gangliosides (designated as G-1, G-2, G-3 and G-4) were purified from bonito fish brain. Ganglioside-1, -2, and -3 migrated above GD1b, below GQ1b, and far below GQ1b on thin-layer chromatography. Ganglioside-4 had the slowest chromatographic mobility and migrated below G-3. The structures of these gangliosides were characterized by overlay analysis with glycolipid-specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G-4 was shown to have the following structure: NeuAc-NeuAc-NeuAc-Galbeta1-3Gal NAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. The antibody A2B5 reacted with these c-series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato- or ganglio-type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c-series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c-series gangliosides.     

Inhibitor of 2',5'-oligoadenylate synthetase induced in human T lymphoblastoid cell line treated with deoxyadenosine, deoxycoformycin and interferon

The effect of deoxyadenosine (dAdo) with deoxycoformycin on the induction of 2',5'-oligoadenylate synthetase by interferon was investigated. After semi-purification through poly(I):poly(C) gel, the activity was similar in control and dAdo-treated cells. However, the activity in the crude extract decreased with rising concentrations of dAdo. On the other hand, the level of 2'-phosphodiesterase, which is also induced by interferon and degrades 2',5'-oligoadenylate, showed no significant change after dAdo treatment. Thus, the crude extract was speculated to contain an inhibitor of 2',5'-oligoadenylate synthetase. Further characterization of the inhibitor revealed that inhibition was not due to dATP accumulation in cells.     

Characterization of human phosphodiesterase 12 and identification of a novel 2'-5' oligoadenylate nuclease - The ectonucleotide pyrophosphatase/phosphodiesterase 1

The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′-5′ oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5′-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2′-5′-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V_max and K_m kinetic parameters for the enzyme. We have also identified a novel 2′-5′-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process.     

The protein phosphatase 2A represents a novel cellular target for hepatitis C virus NS5A protein

It is well established that HCV NS5A protein when expressed in mammalian cells perturbs the extracellular signal regulated kinase (ERK) pathway. The protein serine/threonine phosphatase 2A controls the phosphorylation of numerous proteins involved in cell signaling and one characterized function is the regulation of Ras-Raf mitogen activated protein (MAP) kinase signaling pathways. Our results showed that expression of HCV NS5A protein stimulates phosphatase 2A (PP2A) activity in cells, indicating the relevance of NS5A as a regulator of PP2A in vivo. We found that transient expression of the full length NS5A protein in different cell lines leads to a significant increase of the PP2A activity and this activity is specifically inhibited by the addition of okadaic acid, a PP2A inhibitor, in living cells. Further investigation showed that NS5A protein interacts in vivo and in vitro with the scaffolding A and the catalytic C subunits of PP2A. We propose that HCV NS5A represents a viral PP2A regulatory protein. This is a novel function for the NS5A protein which may have a key role in the ability of the virus to deregulate cell growth and survival.     

Doxifluridine-conjugated 2-5A analog shows strong RNase L activation ability and tumor suppressive effect

RNase L is activated by 2′,5′-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2′-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5′-modified 2-5A analogs with 8-methyladenosine at the 2′-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5′-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Circ_0137287 suppresses cell tumroigenesis and aerobic glycolysis in papillary thyroid carcinoma through miR-183-5p/PPP2R2A axis

Cytotechnology - Circ_0137287 was found to be decreased in papillary thyroid cancer (PTC) tissues and related to aggressive clinicopathologic characteristics. However, the role and mechanism of...     

Interferon-α-(IFN) producing CHO cell lines are resistant to the antiproliferative activity of IFN: A correlation with gene expression

CHO cell lines that constitutively produce the murine interferon-α (IFN-α) subspecies α4 and α6 were constructed. The producer cell lines were protected against viral (vesicular stomatitis virus) infection by the IFN species secreted, but were resistant to the growth inhibitory activity of the IFN species. As compared with α4, the α6 protein displayed a high antiproliferative activity when added to normal CHO cells, which correlates completely with the high antiviral activity of a6 on these cells. Three messenger ribonucleic acid (mRNA) species, which are normally induced in CHO cells by IFN treatment (1–8, 2–5A synthetase, and ISG 15) were constitutively present in CHO producer cell lines. The level of another mRNA (ISG 54), however, was very low in the producer cells as compared with its expression in short-term IFN-treated cells. These data indicate that 1–8, 2–5A synthetase and ISG 15 are not involved in the antigrowth activity of IFN in this system, but rather suggest a function of ISG 54 in this respect.     

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes

Dopamine D2_LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study the existence of D2_L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist ³H-raclopride binding sites and significantly reduced the pK_iH values of the high affinity D2R agonist binding sites in ³H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2_LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists.