Preliminary study of the use of dansyl chloride to determine cyclic-3',5'-AMP in tissues.

A highly sensitive method for the determination of cyclic-3',5'-AMP has been developed which involves the reaction of the substance with dansyl chloride and the subsequent separation of the dansyl-cyclic-3',5'-AMP derivative by thin-layer chromatography. Experiments with standard solutions of 3H-cyclic-3',5'-AMP have shown that there is a direct relationship between the amount of dansyl-3H-cyclic-3',5'-AMP recovered and that dansylated. The procedure is exceedingly sensitive, allowing milligram quantities of material to be analysed for its endogeneous cyclic-3',5'-AMP content. With the use of 14C-adenine as substrate, this method permits the separation of 14C-cyclic-3',5'-AMP formed from the substrate and other 14C-containing compounds, thus allowing the turn-over of cyclic-3'-5'-AMP to be studied. The usefullness of the method is demonstrated by analysing the turn-over and endogenous content of cyclic-3'-5'-AMP in rat nervous tissue.     

Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP), was synthesized via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.     

Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA.

Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.     

PHYTOCHROME VARIATION AND REVERSION IN LEMNA MINOR, L. GIBBA G3 AND L. PAUCICOSTATA 6746

Abstract— Dual wavelength phot00ometry showed an increase of phytochrome in darkness in partially etiolated plants of three heterotrophically cultured Lemna species. In all three species, P_fr formed by a brief illumination decreased in subsequent darkness with half-lives of 2–6 h. In L. minor and L. gibba , dark reversion was observed during the first 8h of darkness; in L. paucicostata , however, only when the plants were in a slightly anaerobic condition. Under continuous illumination, phytochrome decay was not influenced by light intensity. Actinic doses of far red resulted in non-photoreversible changes of absorbance. In material exposed to red for longer than 12 h, these changes increased after preceding actinic doses of red light. This effect may result in erroneous phytochrome estimations.     

DIRECT PHOTO-AFFINITY LABELING OF CYCLIC NUCLEOTIDE BINDING PROTEINS WITH GUANOSINE-3'',5''-MONOPHOSPHATE

Abstract— Radioactivity from [³H]-guanosine-3',5'-monophosphate was shown to become stably linked to proteins in testis extracts during ultraviolet irradiation. Adenosine-3',5'-monophosphate competitively inhibited incorporation into these proteins. Guanosine-3',5'-monophosphate had a lower affinity for binding sites than did adenosine-3',5'-monophosphate. Similar peptides were photo-labeled with [³H]-guanosine-3',5'-monophosphate and [3H]-adenosine-3',5'-monophosphate. Guanosine-3',5'-monophosphate was more efficiently incorporated than was adenosine-3',5'-monophosphate. Extracts from a number of tissues incorporated both cyclic nucleotides photochemically.     

DNA synthesis in cultured human fibroblasts: regulation by 3':5'-cyclic AMP.

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [3H] thymidine incorporation into acid-precipitable material, beginning after 8-12 hr and reaching maximum levels of 16-24 hr. Addition of dibutyryl-3':5'-cyclic AMP (DBcAMP) together with serum inhibited [3H] thymidine incorporation by 75-95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3':5'-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [3H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G1 was inhibited by high concentrations of 3':5'-cyclic AMP.     

碳纳米管-聚(3',4'-亚乙基二氧-2,2':5',2"-三噻吩)复合材料的制备及其电化学性能研究

通过Stille反应合成了3',4'-亚乙基二氧-2,2':5',2"-三噻吩(TET),并以其作为单体,采用化学氧化原位聚合方法在碳纳米管(CNT)的表面包覆新型聚(3',4'-亚乙基二氧.2,2':5',2"-三噻吩)(FTET),制备了PTET-CNT纳米复合材料.通过TEM、SEM和IR对其进行了表征,并利用循环伏安、交流阻抗、恒电流充放电等电化学测试方法,比较研究了复合材料以及碳纳米管在0.1 mol/L四乙基四氟硼酸铵(Et_4NPF_4)的乙腈溶液中的电化学行为.实验结果表明,在电流密度为3 mA/cm~2时,PTET-CNT复合材料的比电容为86 F/g,比原碳纳米管比电容20 F/g提高了3.3倍.基于这种复合材料的电容器的能量密度达到2.02 Wh/kg.     

Elevation of intracellular cyclic adenosine 3',5'-monophosphate levels in macrophages activated with lipopolysaccharide for tumor cell killing.

The macrophage activation for tumor cytotoxicity with lipopolysaccharide (LPS) was remarkably inhibited by adding indomethacin (5 x 10(-6) M) or 10mM LiCl which is known to inhibit adenylate cyclase activity. The tumor cytotoxicity of macrophages inhibited with these agents was recovered by adding dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) (10(-4) or 10(-5) M) and furthermore tumor cell killing activity was augmented as compared with LPS-activated macrophage. Macrophages showed a 5 to 6 times increased intracellular cAMP concentration over the control within 30 min when incubated with LPS. However, the increased intracellular cAMP concentration was decreased by adding LiCl (10 mM). Thus, these findings indicate that there is an important relation between intracellular cAMP concentration and the mechanism of macrophage activation. One can then conclude that at least the initial enhancement of intracellular cAMP was important for tumor cell killing as a signal transmission in macrophage activated by LPS.     

稀土元素对3'-腺嘌呤及3'-鸟嘌呤核苷酸的水解断裂作用

用核磁共振(NMR)波谱、HPLC和化学定磷法研究了稀土元素对3'-腺嘌呤核苷酸(3'-AMP)3及3'-鸟嘌呤核苷酸(3'-GMP)的水解断裂作用, 结果表明: 不同稀土元素对3'位核苷酸断裂有较强的特异性, 在37℃, pH9的条件下, 使3'-AMP断裂为腺嘌呤核苷(A)及无机磷, 使3'-GMP断裂为鸟嘌呤核苷(G)及无机磷, 其断裂作用远大于5'位核苷酸, 并对其水解断裂进行了研究。     

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine)

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine) have been followed by RP HPLC over a wide pH range at 363.2 K in order to elucidate the role of the 2'-hydroxyl group as a hydrogen-bond donor upon departure of the 3'-uridine moiety. Under neutral and basic conditions, guanosyl-(3',3')-uridine undergoes hydroxide ion-catalyzed cleavage (first order in [OH(-)]) of the P-O3' bonds, giving uridine and guanosine 2',3'-cyclic monophosphates, which are subsequently hydrolyzed to a mixture of 2'- and 3'-monophosphates. This bond rupture is 23 times as fast as the corresponding cleavage of the P-O3' bond of guanosyl-(3',3')-(2',5'-di-O-methyluridine) to yield 2',5'-O-dimethyluridine and guanosine 2',3'-cyclic phosphate. Under acidic conditions, where the reactivity differences are smaller, depurination and isomerization compete with the cleavage. The effect of Zn(2+) on the cleavage of the P-O3' bonds of guanosyl-(3',3')-uridine is modest: about 6-fold acceleration was observed at [Zn(2+)] = 5 mmol L(-)(1) and pH 5.6. With guanosyl-(3',3')-(2',5'-di-O-methyluridine) the rate-acceleration effect is greater: a 37-fold acceleration was observed. The mechanisms of the partial reactions, in particular the effects of the 2'-hydroxyl group on the departure of the 3'-linked nucleoside, are discussed.     

MODE OF COACTION OF PHYTOCHROME AND BLUE LIGHT PHOTORECEPTOR IN CONTROL OF HYPOCOTYL ELONGATION

Abstract The rate of hypocotyl longitudinal growth in seedlings of Sesamum indicum L. is strongly inhibited by continuous blue light (cBL)† and slightly by continuous far-red light while continuous red light (cRL) or red light pulses are hardly effective from 60 h after sowing onwards. Between 36 and 60 h after sowing the growth rate responds to red light pulses the effect of which is fully reversible by long wavelength far-red light. When seedlings are kept in cBL for 3 days and then treated with red light hypocotyl growth rate responds strongly. However, RL effectiveness decreases with time after transfer from BL to RL. BL → darkness transfer experiments with different levels of P_fr established at the beginning of darkness show that after a BL pretreatment phytochrome (P_fr) alone is capable of fully controlling growth rate. When white light (WL) is given no BL effect is detectable in weak WL. Only high light fluxes maintain a typical BL growth rate. At medium WL fluxes elongation rate returns gradually to the dark rate. The simplest explanation of the data is that light absorbed by a separate BL photoreceptor is necessary to maintain responsivity to P_fr. With increasing age of the seedlings the requirement for BL increases strongly. On the other hand, brief light pulses—given to demonstrate photoreversibility of phytochrome—remain equally effective provided that responsivity to P_fr exists.     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

EFFECTS OF INORGANIC NITROGEN ON THE RESPONSE OF LEMNA CARBON DIOXIDE OUTPUT TO LIGHT QUALITY AND TIMING*

Abstract —The effects of various light/dark schedules on the time course of CO₂ output by axenic cultures of the short-day plant Lemna perpusilla 6746 differ substantially depending on whether the medium is N-less or contains NH₄ or NO₃ as the sole N source. The steady-state pattern achieved with a daily 1/4 h light pulse in N-less medium is essentially the same whether the light is red or far-red; on NO₃ or NH₄, however, the red and far-red patterns differ in form and suggest the action of a ‘Pfr-hourglass’ timer. In darkness, following either continuous light or entrainment to kh red light daily, CO₂ output oscillates for three or more circadian cycles on NH₄ medium and for at least two on N-less, but damps after a single cycle on NO₃. A schedule of 1/4 h red light every 12 h elicits a 24 h periodicity on NO₃ or NH₄ media and a 12 h periodicity on N-less medium, while a similar far-red schedule elicits a 12 h periodicity on all three. CO₂ output patterns on each of the media respond differently to varying the daily span of light from 1/4 to 6 to 12 h. These results are probably due to differential effects of changing N status on the proportion of total C O₂ arising from various metabolic reactions. They suggest that, rather than being a simple, unitary indicator, CO₂ output can be made to reflect different processes on different media, increasing its value as a real-time indicator of events underlying photoperiodism.     

The synthesis and photoactivated cytotoxicity of novel 5-phenyl-3- （2,2＇：5＇,2″-terthien-5-yl）-4,5-dihydro-lH-pyrazolines

A series of novel 5-phenyl-3-（2,2＇：5＇,2″-terthien-5-yl）-4,5-dihydro-lH-pyrazolines were synthesized in this report. Their photoactivated cytotoxicities on the 5podoptera litura （SL） cell line were evaluated using the MTT method. It was noticed that the inhibitory activities of all the conjugates was enhanced when irradiated with UV-A light. Compounds 4, 6 and 8 were found to be most effective with inhibition rates of 88.1%, 88.0%, and 90,5%, respectively, For compound B, the inhibition rate value was only slightly enhanced under the irradiation treatment （78.3%） compared to the dark treatment （74.8%）. The relationship analysis between structure and activity showed that the middle thiophene ring played an important role on the inhibitory activities. It was shown that these compounds could be the potential candidates for new photoactivated pesticides.     

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated cells.     

Novel benzyl rearrangements in electrospray ionization multistage tandem mass spectra of benzyl 2',3'- didehydro-2',3'-dideoxythymidin-5'-yl H-phosphonate

Several alkyl 2',3'-didehydro-2',3'-dideoxythymidin-5'-yl H-phosphonates were synthesized and analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Two kinds of novel benzyl rearrangement reactions were observed in ESI - MS(2) of [M + H](+), [M + Na](+) and [M + K](+) of benzyl 2',3'-didehydro-2',3'-dideoxythymidin-5' yl H-phosphonate. Results from tandem mass spectrometry, high-resolution mass spectrometry and control experiments showed that the benzyl migration could undergo a four-membered cyclic rearrangement reaction, and benzyl was essential in the process.     

Hydrolytic stability of 2',3'-O-methyleneadenos-5'-yl 2',5'-di-O-methylurid-3'-yl 5'-O-methylurid-3'(2')-yl phosphate: implications to feasibility of existence of phosphate-branched RNA under physiological conditions

Hydrolytic reactions of 2',3'-O-methyleneadenos-5'-yl 2',5'-di-O-methylurid-3'-yl 5'-O-methylurid-3'(2')-yl phosphate (1a,b) have been followed by RP-HPLC over a wide pH range to evaluate the feasibility of occurrence of phosphate-branched RNA under physiological conditions. At pH <2, where the decomposition of is first order in [H3O+], the P-O5' bond is cleaved 1.5 times as rapidly as the P-O3' bond. Under these conditions, the reaction probably proceeds by an attack of the 2'-OH on the phosphotriester monocation. Over a relatively wide range from pH 2 to 5, the hydrolysis is pH-independent, referring to rapid initial deprotonation of the attacking 2'-OH followed by general acid catalyzed departure of the leaving nucleoside. The P-O5' bond is cleaved 3 times as rapidly as the P-O3' bond. At pH 6, the reaction becomes first order in [HO-], consistent with an attack of the 2'-oxyanion on neutral phosphate. The product distribution is gradually inversed: in 10 mmol L(-1) aqueous sodium hydroxide, cleavage of the P-O3' bond is favored over P-O5' by a factor of 7.3. The results of the present study suggest that the half-life for the cleavage of under physiological conditions is only 100 s. Even at pH 2, where is most stable, the half-life for its cleavage is less than one hour and the isomerization between and is even more rapid than cleavage. The mechanisms of the partial reactions are discussed.     

PHOTOPRODUCTS OF BROMOURACIL-LABELED DNA AND THE STRUCTURE OF 5-BROMODEOXYURIDYLYL-(3' → 5')-THYMIDINE PHOTOPRODUCT

Abstract— –An attempt was made to identify some of the ultraviolet (u.v.) photoproducts of 5-bromouracil-labeled DNA (BrU-DNA). Two synthetic dinucleotides, 5-bromodeoxyuridylyl-(3' →5 ')-thymidine (BrdUpT) and 5-bromodeoxyuridylyl-(3' → 5')-deoxycytidine (BrdUpdC), were prepared. Each gave a single u.v. photoproduct which in turn gave a single acid hydrolysis product. 2-¹⁴C-BrU-DNA. prepared from E. coli B3, was irradiated (275–280 nm), hydrolyzed, and paper chromatographed in four systems. Comparison with the two synthetic photoproducts showed that if present at all, BrdUpT and BrdUpdC photoproducts could account for no more than 10 and 3.5 per cent respectively of the total photoproducts. At 55 per cent conversion of BrU into photoproducts, the major ¹⁴C-photoproduct was uracil (78 per cent); the remaining 22 per cent was made up of at least six products, three of which were reversed by 232 nm irradiation.
The debrominated cyclobutane structure proposed by Haug for BrdUpT photoproduct has been shown to be incorrect. It was found to contain one atom of bromine per molecule. On the basis of nuclear magnetic resonance and u.v. spectra, two possible structures are proposed for the photoproduct, each containing an eight-membered ring.     

Hydrolysis of 2',3'-O-methyleneadenos-5'-yl bis(2',5'-di-O-methylurid-3'-yl) phosphate, a sugar O-alkylated trinucleoside 3',3',5'-monophosphate: implications for the mechanism of large ribozymes

Hydrolytic reactions of 2',3'-O-methyleneadenos-5'-yl bis(2',5'-di-O-methylurid-3'-yl) phosphate (1), a sugar O-alkylated trinucleoside 3',3',5'-monophosphate, have been followed by RP HPLC over a wide pH range. Under neutral and mildly acidic conditions, the only reaction observed was a pH-independent cleavage of the O-C5' bond of the 5'-linked nucleoside. Under more alkaline conditions nucleophilic attack by hydroxide ion starts to compete. The reaction is first order in [OH(-)] and becomes predominant at pH 10. Each of the 3'-linked nucleosides is displaced 2.9 times as readily as the 5'-linked one. To determine the beta(lg) value for the hydroxide ion catalyzed hydrolysis of 1, two diesters (2a,b) having 2',3'-O-methyleneadenosine (7) and 2',5'-di-O-methyluridine (4) as leaving groups were hydrolyzed under alkaline conditions. Since the beta(lg) value for this reaction is known, DeltapK(a) between 4 and 7 could be calculated. The beta(lg) for the hydrolysis of 1 was estimated to be -0.5 with use of this information. The mechanisms of the partial reactions and the role of leaving group properties in ribozyme reactions of large ribozymes are discussed.