New Phosphonoformic Acid Derivatives of 3′-Azido-3′-Deoxythymidine

New 5-alkyl ethoxy- and aminocarbonylphosphonates of 3-azido-3-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.     

Genetic control of chalcone-flavanone isomerase activity in Callistephus chinensis

A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2,4,6-tetrahydroxy-chalcone-2-glucoside (isosalipurposide) in blossoms of Callistephus chinesis (L.) Nees, whereas in geno-types with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalcone-flavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and there-with furnishes the substrate for the further reactions in the flavonoid biosynthesis.Abbreviations EGME ethylene glycol monomethyl ether - HOAc acetic acid - MeOH methanol - PVP polyvinylpyrrolidone - TBA tert. butanol-acetic acid-water, 3:1:1 - TLC thin-layer chromatography     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

Calmodulin genes in zebrafish (revisited)

Calmodulin (CaM), a ubiquitous protein, ancestral in early eukaryotes, regulates a large number of physiologically important functions by activating other proteins, some of them enzymes, usually in response to changes in the local concentration of calcium ions. Invertebrates possess one gene that codes for CaM. Among vertebrates, mammals display three genes that code for a 100% identical CaM molecule, while for zebra fishes etc., a non-mammalian vertebrate, we reported earlier the existence of four such genes. The number of multiple genes coding for a 100% identical CaM molecule present in the zebra fish genome, however, is corrected here, from the four, as previously suggested, to six (alpha, alpha2, beta, beta2, gamma and gamma 2). Identification of each of these genes is readily achieved upon examination of the characteristic 5 and 3 UTRs within their respective mRNAs even though we do not know at present what role these UTRs might play. A scanning of the 3 UTRs for short homology elements among the six genes (and a comparison with the human type I, II, and III CaM 3 UTRs) also suggests that duplication processes for three genes resulted in the formation of six such genes. As they become available, the promoter regions for these six genes should be scanned for possible identification of putative regulatory elements if we are to understand the need for the uniquely rigid evolutionary maintenance of these six genes. A comparison of the promoter regions for the beta and beta 2 genes is presented in this paper. A few common short homologous elements appear to be retained in these generally highly variant two regions, but conclusions about differential expression controls must be delayed until the promoter regions for all the other CaM genes have been examined.     

The effect of ATP on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) regeneration from nodal explants: association with α-tocopherol,H<Subscript>2</Subscript>O<Subscript>2</Subscript> and size of culture vessel

We investigated the effect of adenosine 5-triphosphate (ATP), -tocopherol and H₂0₂ on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants. The effect of the size of the liquid culture vessel (250-ml flask vs. 2.5-l airlift bioreactor) was also evaluated. The addition of ATP alone caused a significant increase in the number of branches (internodes) and in internodal length, but also a reduction of leaf number, compared to control cultures. It also increased significantly the accumulation of NO₃^– and K⁺ . The application of ATP + -tocopherol was associated with a significant increase in bud, internodal, leaf and petiole number, internodal, petiole and root length, as well as plant fresh weight, but reduced PO₄^3– and Ca²⁺ accumulation. The combined application of ATP + -tocopherol + H₂O₂ was associated with maximum petiole length and increased plant fresh weight but reduced Ca²⁺ accumulation. Compared to all other treatments, application of ATP + -tocopherol in bioreactor-incubated cultures produced significantly larger plants, with an increased bud number, internode lenght and soluble carbohydrate concentration, but also with a reduced fresh weight, root length and reduced NO₃^– and PO₄^3– and Ca²⁺ concentrations. These effects were associated with changes in the redox status of the regenerants, as well as dehydrogenase and peroxidase activities. The perspective for an application of ATP and antioxidants as novel, cost-efficient growth regulators in the micropropagation of this commercially important vegetable species is discussed.     

Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in a cyclic 3′:5′ adenosine monophosphate-dependent protein kinase mutant of Neurospora crassa

The possibility that plant growth regulators may relate to a cyclic 3:5 adenosine monophosphate (cAMP)-dependent protein kinase through the control of cAMP level in the conidial germination process of Neurospora crassa was examined using a cAPM-dependent protein kinase mutant (cpk mutant) which is thought to be cAMP-independent because of defect in the regulatory subunit of cAMP-dependent protein kinase. IAA, 2,4-D and GA₃ promoted conidial germination and elongation of young hyphae in the mutant as well as in the wild-type. The result suggests that the effects of auxin and gibberellin on germination and hyphal elongation are not mediated by cAMP.     

Isolation of trace amounts of 3′,4′-dehydro-4′-deoxydothistromin from peanut plants naturally infected with Cercospora personata

Trace amounts of the anthraquinonoid toxic metabolite, 3,4-dehydro-4-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and Chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Effects of d-propranolol and estradiol on the cervicovaginal epithelium

Summary The cervicovaginal epithelium of neonatal mice produces a material with specific antigenic properties (CVA) and this material is produced in increased amounts after estradiol treatment. Using a cytochemical method, estradiol treatment was shown to result in an increase of adenylate cyclase activity in the same epithelium.When d-propranolol is injected together with estradiol, the increase in CVA is inhibited, while the hormone-induced proliferation of epithelial cells is not influenced. When adenylate cyclase activity is studied under identical conditions, the estradiol-promoted increase in enzyme activity is largely counteracted by d-propranolol. These findings would suggest that Adenosine 35-cyclic monophosphate (cAMP) has a role in some, but not all, estradiol-mediated effects in the neonatal cervicovaginal epithelium.This work was supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society (Landsforeningen mot Kreft)     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Isolation and characterization of the ecto-5′-nucleotidase from a rat glioblastoma cell line

Summary 5-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mol AMP-min^–1-mg^–1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5-nucleotidase presents optimum activity at pH 7.8–8.1 either in the presence or in the absence of Me²⁺. A linear Arrhenius plot is observed in the 25–46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn²⁺, Mn²⁺, and Co²⁺. The hydrolysis of nucleosides 5-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.     

N,N′-carbonyldiimidazole-induced diketopiperazine formation in aqueous solution in the presence of adenosine-5′-monophosphate

Summary 3(2)-O-glycyl-adenosine-5-monophosphate is an intermediate in the conversion of N-[imidazolyl-(1)-carbonyl]-glycine to diketopiperazine in the presence of adenosine-5-monophosphate. The significance of these observations to prebiotic chemistry is discussed.Abbreviations AMP adenosine-5-monophosphate - A adenosine     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Antimutagenic Role of Autonomous 3′ → 5′-Exonucleases

Original and published data on the antimutagenic role of autonomous 3 5-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but are often involved in replicative complexes. AE overproduction in bacterial cells is accompanied by a sharp suppression of mutagenesis, whereas AE inactivation in bacteria and higher fungi results in the increase in mutation rates by two to three orders of magnitude. The combined action of AE and DNA polymerases substantially improves the fidelity of their functioning in vitro. The fidelity of nuclease-free DNA polymerases and increases by two to three orders of magnitude in the presence of AE. The fidelity of moderately processive DNA polymerase I increases by two orders of magnitude, and that of highly processive DNA polymerase increases by a factor of 5–10, although both these polymerases possess their own 3 5-exonucleolytic activity. In biochemical experiments, AE was shown to participate directly in the correction of errors made by DNA polymerase I. The presence of AE in multienzyme DNA polymerase complexes increases their fidelity by a factor of 5–10. A model of extrinsic proofreading by AE in DNA biosynthesis is proposed. An investigation of thirty objects from all three kingdoms of life (from archaea and bacteria to mammals, including humans) has shown that AE account for 30–90% of the total cellular 3 5-exonucleolytic activity. Therefore, AE increase significantly the intracellular ratio of 3 5-exonuclease to DNA polymerase activities in a wide phylogenetic variety of species, which always leads to the increasing fidelity of DNA biosynthesis.     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇