Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Histochemical method for localization of cyclic 3′, 5′-nucleotide phosphodiesterase

Summary A histochemical method has been described for demonstration of cyclic 3, 5-nucleotide phosphodiesterase in tissues. 5-AMP is formed due to splitting of substrate cyclic 3, 5-AMP by cyclic 3, 5-AMPase. The 5-AMP is split into adenosine and phosphate by the 5-nucleotidase from added snake venom. Endogenous tissue 5-nucleotidase would contribute to this activity. The phosphate was in turn visualized by conversion to the lead salt in the presence of lead acetate and finally into brownish-black lead sulphide by treatment with yellow ammonia sulphide. Control studies with and without substrate and snake venom, as well as inhibition by theophylline, indicate the test to be specific for cyclic 3, 5-AMPase.In the eye the conjunctiva, ciliary process, choroid and retina all showed strongly positive activity. In the kidney the proximal and distal tubules both ascending and descending and the loop of Henle show strongly positive activity — the rest of the elements being negative. The cardiac and skeletal muscle exhibited very little positive activity. The liver showed only mildly positive activity. The villi of the small intestine showed strongly positive activity at the apical part of the cells. Neurons showed very little positive activity in either the cerebral cortex or the cerebellum. On the other hand, the molecular layer in the cerebellum and the plexiform layer of the cerebral cortex showed strongly positive activity. The significance of these findings are briefly discussed. T. R. Shanthaveerappa — in previous publications.     

Cyclic nucleotide phosphodiesterases of the renal cortex

Summary Cyclic nucleotide phosphodiesterase in the basal-lateral segment of plasma membranes from proximal tubule cells of the rabbit renal cortex was studied and compared to that in the brush border segment of the plasma membrane. Both adenosine 3,5-monophosphate and guanosine 3,5-monophosphate were hydrolyzed by the basal-lateral membrane, but activity varied differently with the two substrates in a complex concentration-dependent manner. Activity with adenosine 3,5-monophosphate was greater than, equal to, or less than with guanosine 3,5-monophosphate, at concentrations of 1000, 100, and 10 to 1 m, respectively. Basal-lateral membrane phosphodiesterase activities at 1 and 500 m substrate exhibited differential responses to pH, metals, heat, and a heat stable inhibitor. Stimulation by guanosine 3,5-monophosphate and inosine 3,5-monophosphate of adenosine 3,5-monophosphate hydrolysis was found in basal-lateral but not in brush border membranes. This stimulation was potentiated by ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid and ethylenediaminetetraacetate, inhibited by Triton X-100, and totally blocked by Zn²⁺. The findings indicate that multiple forms of phosphodiesterase are present in the basal-lateral segment and these differ from the activities in the brush border region of the plasma membrane. The characteristics of (i) allosteric, guanosine 3,5-monophosphate-sensitivity of adensoine 3,5-monophosphate phosphodiesterase, and (ii) relatively high guanosine 3,5-monophosphate phosphodiesterase activity, in basal-lateral membranes, which are also enriched in adenylate and guanylate cyclase, suggest an important physiological role for these phosphodiesterases in the regulation of net production of cyclic nucleotides in the renal cortex.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Amino acid activation with adenosine 5′-phosphorimidazolide

Summary Amino acids are activated by reaction with adenosine 5-phosphorimidazolide in aqueous imidazole buffers. If adenosine 5-(O-methylphosphate), an analogue of the 3-terminus of t-RNA is present, 2(3)-O-aminoacyladenosine 5-(O-methylphosphate) is formed. Fifteen percent of this compound accumulated at pH 5.8, but less was formed at higher pHs. The highest efficiency of utilization of ImpA attained in our experiments was about 24%. Analogous reactions occured with several other amino acids, including a number that have functional side-chains.Abbreviations pA adenosine 5-monophosphate - MepA adenosine-5-(O-methylphosphate) - ImpA adenosine-5-phosphorimidazolide - A adenosine - MepA-ala 2(3)-O-alanyl-adenosine-5-(O-methylphosphate) - ala-N-pA adenylyl-(5 N)-alanine - ImH imidazole - DKP diketopiperazine     

Mineral catalysis of the formation of dimers of 5′-AMP in aqueous solution: The possible role of montmorillonite clays in the prebiotic synthesis of RNA

The reaction of the 5-AMP with water soluble carbodiimide (EDAC) in the presence of Na⁺-montmorillonite 22A results in the formation of 2,5-(pA)₂ (18.9%), 3,5-(pA)₂ (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2,5-(pA)₂ (15.5%), 3,5-(pA)₂ (3.7%) and AppA (14.9%). The 3,5-cyclic dinucleotide, 3,5-c(pA)₂, is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. Products which contain the phophodiester bond are formed at different ionic strengths, pH and temperatures using Na⁺-montmorillonite. Phosphodiester bond formation was not observed when Cu²⁺-montmorillonite was used or when DISN was used in the place of EDAC. The extent catalysis of phophodiester bond formation varied with the particular clay mineral used. Those Na⁺-clays which bind 5-AMP more strongly are better catalysts. Cu²⁺-montmorillonite, which binds 5-AMP strongly, exhibits no catalytic activity.     

2′(3′),5′-ADP inhibits initiation-dependent protein synthesis in a cell-free system from Ehrlich ascites tumor cells

When tested in a poly(U)-dependent polyphenylalanine synthesizing system and in a postnuclear supernatant, both derived from Ehrlich ascites tumor cells, 2(3),5-ADP did not affect chain elongation of polypeptide synthesis. In a cell-free system which was dependent on initiation and programmed by natural mRNA, however, the amino acid incorporating activity was suppressed to about 10% of the control in the presence of 1 mM 2(3),5-ADP. The inhibitor was shown not to interfere with the attachment of poly(U) to the small ribosomal subunit and with the formation of mRNA-80S ribosome complexes in a complete protein synthesizing system. The subsequent attachment of a 40S ribosomal subunit to the mRNA-80S ribosome complex and the formation of polysomes, however, was depressed by the inhibitor. The experimental results suggest that 2(3),5-ADP inhibits initiation-dependent protein synthesis between monosome formation and the formation of the first peptide bond(s).     

Cleavage of nucleotides by Ce4+ and the lanthanide metal complexes

The cleavage of adenosine-5-monophosphate (5-AMP) and guanosine-5-monophosphate (5-GMP) by Ce⁴⁺ and lanthanide complex of 2-carboxyethylgermanium sesquioxide (Ge-132) in acidic and near neutral conditions was investigated by NMR , HPLC and measuring the liberated inorganic phosphate at 37°C and 50°C. The results showed that 5-GMP and 5-AMP was converted to guanine (G), 5-monophosphate (depurination of 5-GMP), ribose (depurination and dephosphorylation of 5-GMP), phosphate and adenine (A), 5-monophosphate (depurination of 5-AMP), ribose (depurination and dephosphorylation of 5-AMP), phosphate respectively by Ce⁴⁺. In presence of lanthanide complexes, 5-GMP and 5-AMP were converted to guanosine (Guo) and phosphate and adenosine (Ado) and phosphate respectively. The mechanism of cleaving 5-GMP and 5-AMP is hydrolytic scission     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

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     

Phagostimulation of the mediterranean fruit fly, Ceratitis capitata by ribonucleotides and related compounds

Females of the medfly, Ceratitis capitata, prefer sucrose solutions containing ribonucleotides to sucrose solutions without them. The order of preference for the nucleotides was: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP=2 & 3GMP=RP>3AMP.2AMP, guanosine, inosine, adenine and 5TMP produced no significant stimulation. Females sterilized by irradiation showed reduced attraction to 5GMP as compared to non-irradiated females.Optimal molecular configuration for phagostimulation includes: phosphorylation at the 5 position of the ribose, free hydroxyl groups at 2 and 3 on the ribose, and an NH₂ group at the 2 position of the aromatic ring of purine.It is proposed that the 5GMP in yeast hydrolyzate can be used as a measure of the suitability of the hydrolyzate as a bait.

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Résumé La femelle de la mouche méditerranéenne des fruits, Ceratitis capitata, préfère les solutions de sucrose contenant des ribonucléotides aux simples solutions de sucrose. Lórdre de préférence pour les nucléotides est le suivant: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP =2 & 3GMP=RP>3AMP.Le 2AMP, la guanosine, l'inosine, l'adénine et le 5TMP provoquent une stimulation significative. Les femelles montrent aprés stérilisation par irradiation une attirance réduite pour le 5GMP par comparaison avec les femelles non-irradiées.La configuration moléculaire optimale pour la phagostimulation comprend: la phosphorylation en position 5 du ribose; des groupes hydroxyles libres en 2 et 3 sur le ribose; et un groupe NH₂ en position 2 sur le noyau aromatique.Nous proposons que le 5GMP dans l'hydrolysat de levure puisse être utilisé pour mesurer la capacité de l'hydrolysat comme appât.

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Abbreviations 5AMP Adenosine 5-monophosphate - 3AMP Adenosine 3-monophosphate - 2AMP Adenosine 2-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - 5GMP Guanosine 5-monophosphate - 2GMP Guanosine 2-monophosphate - 3GMP Guanosine 3-monophosphate - dGMP 2-deoxyguanosine 5-monophosphate - GDP Guanosine 5-diphosphate - GTP Guanosine 5-triphosphate - 5IMP Inosine 5-monophosphate - IDP Inosine 5-diphosphate - ITP Inosine 5-triphosphate - 5XMP Xanthosine 5-monophosphate - 5CMP Cytidine 5-monophosphate - dCMP 2 deoxycytidine 5-monophosphate - CTP Cytidine 5-triphosphate - 5UMP Uridine 5-monophosphate - 5TMP Thymidine 5-monophosphate - RP Ribose 5 monophosphate     

Oligomerization reactions of ribonucleotides: The reaction of the 5′ -phosphorimidazolide of adenosine with diadenosine pyrophosphate on montmorillonite and other minerals

The reaction of the 5 -phosphorimidazolide of adenosine (5-ImpA) with diadenosine pyrophosphate (A⁵ppA) in the presence of Na⁺-montmorillonite in aqueous, pH 8 solution results in the regiospecific formation of A⁵ppA³pA and A⁵ppA³pA³ pA. The formation of oligomers of general structure (pA)n decreases in the presence of A⁵ppA. A⁵ppA³pA is the principal reaction product when a 1:1 ratio of ImpA and A⁵ppA is used. The yield of A⁵ppA³pA³pA is optimal when 9:1 or 4:1 ratios of ImpA: A⁵ppA are used. The overall regiospecificity of formation of 3,5-links is about 80%. The reaction between ImpA and A⁵ppA on montmorillonite differs from the self-condensation of ImpA in that it proceeds in the absence of Mg²⁺ and there are only small differences in oligomer yields when Na⁺, Li⁺ Ca²⁺, and NH ₄ ⁺ are the exchangeable cations on the montmorillonite. The reaction is inhibited by 0.4 M imidazole but the inhibition is suppressed with 0.4 M Mg²⁺. Little or no phosphodiester bond formation was observed with Mg²⁺- or Al³⁺-montmorillonite. Montmorillonites other than 22A and Volclay exhibited no catalysis for the formation of adducts between ImpA and A⁵ppA and no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, or sepiolite.     

RNA- synthesis inAcetabularia

Summary Incorporation of precursors of RNA into the enucleated cells ofAcetabularia mediterranea andPolyphysa cliftonii has been studied under conditions which exclude the possibility of errors due to contamination of the preparations by nuclei, nuclear debris or microorganisms.Radioactive RNA has been isolated from the chloroplast, mitochondrial and supernatant cytoplasm fractions of nucleated and enucleated cells. Sucrose density gradient centrifugation of the isolated labelled RNA produces a sedimentation profile of radioactivity which is similar to that of RNA isolated fromE. coli ribosomes.Anion exchange chromatography of alkaline hydrolysates of the 23 s and 16 s ribosomal RNA fractions shows incorporation of labelled uracil into RNA in the form of 2(3)-UMP and 2(3)-CMP. Labelled guanosine is incorporated only as 2(3)-GMP.A slowly sedimenting radioactivity peak has been chromatographed on a Hershey column and found to correspond to cold t-RNA.Some of the results were presented at the Symposium on Some Biological Systems at the Molecular Level organized by the International Organization for Pure and Applied Biophysics in Naples in September, 1965.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Influence of cyclic nucleotides on amphibian ectoderm

Summary Isolated amphibian (Triturus alpestris) gastrula ectoderm was treated with cyclic nucleotides for 24 h and cultured up to 12 days. Explants treated with$cyclic N⁶-Monobutyryl-adenosine-35-monophosphate, cyclic Dibutyryladenosine-35-monophosphate and cyclic Dibutyrylguanosine-35-monophosphate in a concentration of 10^–3 and 10^–5 M did not differentiate into mesoderm- or endoderm-derived tissues. The number of explants with small neural and neuroid structures did not exceed the percentage found in the control series. Inductions could also not be obtained when ectoderm was dissociated prior to the treatment with cyclic nucleotides, or when theophylline (which inhibits phosphodiesterase) was added to the culture medium. The results are discussed with regard to the possible mode of action of the vegetalizing factor.     

Species diversities analyzed by density and cover in an early volcanic succession

To evaluate alpha diversities, various variables such as density, cover, volume, and weight have been used. However, density is often a distinct variable from the remaining three. To clarify differences in diversity measured by those two kinds of variables, the data collected in fourteen 2×5 m permanently-marked plots on Mount Usu, Japan, which erupted during 1977 and 1978 in growing seasons from 1983 to 1989 was analyzed, using Shannon's species diversity (H) that is represented as a result of combination of species richness and evenness (J). H and J were evaluated by density (density H and J) and cover (cover H and J). Cover H and J were significantly lower than density H and J, indicating that cover H has different characteristics from density H. Those differences are due to differences in evenness, because species richness is the same. The rank orders of species density are different from those of cover. The predominance of a few perennial herbs greatly decreases cover evenness, while seedling establishment success influences density evenness. Therefore, I propose that, during the early stages of succession on harsh environments such as volcanoes, density diversity represents seedling establishment success rate while cover diversity expresses vegetative reproduction success rate.