Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate

A series of adenosine cyclic 3',5'-phosphate (cAMP) derivatives containing modifications or substitutions in either the 2',3',4', or 5' position or the phosphate were examined for their abilities to activate type I isozymes of cAMP-dependent protein kinase (PK I) from rabbit or porcine skeletal muscle and type II isozymes of cAMP-dependent protein kinase (PK II) from bovine brain and heart. The studies revealed that the activation of both PK I and PK II isozymes requires a 2'-hydroxyl group in the ribo configuration, a 3' oxygen in the ribo configuration, and a charged cyclic phosphate. The two isozymes appeared to differ in those portions of their respective cAMP-binding sites that are adjacent to the 4' position of the ribose ring and the 3' position, 5' position, and phosphate portion of the cyclic phosphate ring.     

Stimulation of cholinergic receptors and changes in cyclic nucleotide levels in the cerebral cortex of the guinea pig

The effect of Atropine and Physostigmine on 3'5' AMP and 3'5' GMP content was investigated in slices of guinea-pig cerebral cortex maintained at rest or electrically-stimulated. Atropine and Physostigmine did not modify either the basal content or the electrically-evoked increase of 3'5' AMP and 3'5' GMP. On the contrary, Betanechol 25 micro M significantly increased 3'5' GMP and 3'5' AMP content in slices kept at rest. The effect was abolished by Atropine 1,5 x 10(-7) M and d-tubocurarine 7 x 10(-6) M, respectively.     

A conformational study of adenylyl-(3'',5'')-adenosine and adenylyl-(2'',5'')-adenosine in aqueous solution by carbon-13 magnetic resonance spectroscopy.

The solution conformation of adenylyl-(3',5')-adenosine and adenylyl-(2',5')-adenosine in both the stacked and unstacked states was studied by carbon-13 magnetic resonance spectroscopy. Large chemical shift differences between the base carbons in the dimers and those in the corresponding monomers are attributed in part to the influence of base-base interaction. Carbon-phosphorus couplings across three bonds revealed the preferred populations for certain backbone rotamers, demonstrating that significant changes in conformation about the "c(3')-O and C(5')-O bonds do not occur in the temperature or salt-induced unstacking of adenylyl-(3',5')-adenosine. However, rotations about the C(2')-O and C(5')-O bonds occur in the temperature-mediated unstacking of adenylyl-(2',5')-adenosine.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.     

N6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5'-C-C-G-G-3') or seven HhaI (5'-G-C-G-C-3') sites upstream from the 3' located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5' located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3' located). Deoxycytidine methylations at two AluI (5'-A-G-C-T-3') sites downstream from the 5' located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5'-G-A-A-T-T-C-3') or one TaqI (5'-T-C-G-A-3') sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5' located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5'-G-A-T-C-3') site, which is located 127 nucleotides downstream from the 5' located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N6-methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Heterogeneous nuclear RNA promotes synthesis of (2'',5'')oligoadenylate and is cleaved by the (2'',5'')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.     

Chemical structures of mono-, di-, tri-, and tetraglycosyl glycerides in rice bran.

1. Monoglycosyl monoglyceride, mono-, di-, tri- and tetraglycosyl diglycerides were isolated from rice bran and characterized for their chemical structures. 2. Monoglycosyl monoglycerides were characterized as Gal(beta 1' leads to 3)-1- or 2-monoacyl-sn-glycerol and Glc(beta 1' leads to 3)-1- or 2-monoacyl-sn-glycerol. 3. The structures of monoglycosyl diglyceride were Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol and Glc(beta 1' leads to 3)-1,2diacyl-sn-glycerol. Epimeric separation of the galactosyl and glucosyl glycerides was for the first time achieved by thin-layer chromatography. 4. The main diglycosyl diglyceride was shown to be Gal(alpha 1' leads to 6')-Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol. 5. The major structure of triglycosyl diglyceride was characterized as Gal(alpha 1' leads to 6')-Gal(alpha 1' leads to 6')-Gal(beta 1' leads to 3)-1,2-diacyl-sn-glycerol. 6. The representative structure of tetraglycosyl diglyceride was for the first time established as Gal(alpha 1' leads to 6')-Gal(alpha 1' leads to 6')-Gal(a-pha 1' leads to 6')-Gal(beta1' leads to 3)-1,2-diacyl-sn-glycerol.     

The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').     

A simple procedure for synthesis of diastereoisomers of thymidine cyclic 3',5'-phosphate derivatives

Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.     

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.     

Enhancing the stability of oligonucleotide duplexes. The effect of adding 1-(2'-deoxy-beta-d-ribofuranosyl)-5-nitroindole

We studied the properties of DNA duplexes containing 5-nitroindole (N) in one of the chains. We synthesized 8-membered oligos with N at the 5' or at the 3' end: 5'-d(NXGACCGTC)-3' or 5'-d(GACCGTCXN)-3', where X is one of the four natural bases, making all four kinds of oligos with and without N. We also prepared 11-membered oligos complementary to the above octanucleotides: 5'-d(TGACGGTCYZT)-3' and 5'-d(TZYGACGGTCT)-3', where Y and Z are A, G, C, or T. The stability of duplexes obtained with these oligos was assessed by melting, and the thermodynamic parameters delta H, delta S, and Tm were calculated. Comparison of the melting curves for modified and nonmodified duplexes demonstrated that the presence of N at the 5' end of one chain raises the Tm by 6.6 degrees C on average; if N is at the 3' end of the same chain, the Tm increases by about 3 degrees C.     

2''5'' oligoadenylate synthetase, an interferon induced enzyme: direct assay methods for the products, 2''5'' oligoadenylates and 2''5'' co-oligonucleotides.

The interferon induced enzyme 2'5' oligoadenylate synthetase produces 2'5' pppA(pA)n the first discovered natural nucleotide with a 2'5' linkage. We describe a direct assay of this enzyme based on separation by thin layer chromatography (TLC) of the substrate ATP and the products 2'5' pppA(pA)n (n larger than or equal to 1). This technique presents obvious advantages compared to the currently used methods. Moreover the enzyme uses other nucleotides as substrates forming co-oligonucleotides 2'5 pppA(pA)n pN (N = U,G,C,dA,dG,dT and dC). Additional procedures are described using different developing solvent systems for the separation of the core-2'5' oligonucleotides (2'5' A(pA)npN) containing AMP-residues entirely and those with another nucleotide at the 2' end.     

A proton magnetic resonance investigation of the glycosyl torsion angle of uracil nucleosides and nucleotides.

The use of line-shape decomposition techniques permitted the small 5-bond (5-J51') and 4-bond (4-J61') proton-proton coupling constants of a series of uracil nucleosides and nucleotides to be determined accurately. From an analysis of these coupling constants we have determined that the uracil base is in a predominantly anti conformation in aqueous solution and the mean position is not substantially altered by phosphate substitution at the 2', 3', or 5' positions, by changing the furanose stereochemistry from a ribose to a deoxyribose or an arabinose, or by an increase in temperature of 43 degree C.     

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.     

Uridine recognition motifs of human equilibrative nucleoside transporters 1 and 2 produced in Saccharomyces cerevisiae

The sugar moiety of nucleosides has been shown to play a major role in permeant-transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3'). The C(2')-OH at uridine was a structural determinant for uridine-hENT1, but not for uridine-hENT2, interactions. Both transporters were sensitive to modifications at C(5') and hENT2 displayed more tolerance to removal of C(5')-OH than hENT1; addition of an O-methyl group at C(5') greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3')-OH and moderate interactions with C(2')-OH and C(5')-OH of uridine, whereas hENT2 formed strong interactions with C(3')-OH, weak interactions with C(5')-OH, and no interaction with C(2')-OH.     

2,2',6,6'-Tetrachlorobiphenyl is estrogenic in vitro and in vivo

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho-substituted PCB, 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-TeCB). This PCB was chosen as the prototypical ortho-substituted PCB to test the hypothesis that ortho-substitution of a PCB with no para- or meta-chlorine-substitutions results in enhanced estrogenic activity. The results indicate that 2,2',6,6'-TeCB is estrogenic both in vitro, in the MCF-7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 microM 2,2',6,6'-TeCB to the medium of MCF-7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2',6,6'-TeCB or a metabolite is acting through an ER-dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2',6,6'-TeCB does not bind rhERalpha or rhERbeta. A metabolite of 2,2',6,6'-TeCB, 2,2',6,6'-tetrachloro-4-biphenylol (4-OH-2,6,2',6'-TCB), does bind rhERalpha and rhERbeta and is also 10-fold more estrogenic than 2,2',6,6'-TeCB in the MCF-7 focus assay; however, this metabolite is not detected in the medium of MCF-7 cultures exposed to 2,2',6,6'-TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2',6,6'-TeCB binding to the ER, 2) 2,2',6,6'-TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER.