Binding of 8-bromoguanylic acid to ribonuclease T1 as studied by absorption and circular dichroism spectroscopy

8-Bromoguanosine 2'- and 3'-phosphates have been shown to bind to RNase T1 with the same affinity as the corresponding guanosine phosphates, inducing difference absorption and circular dichroism spectra similar to those induced by the guanosine phosphates. Since the brominated ligands have reduced electron density on N-7 of the guanine ring and syn-fixed conformation due to a bulky, electron-withdrawing Br substituent on C-8, the difference spectra are not attributable to the protonation on N-7 and to the restriction of the ligand to syn-conformation as proposed previously.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Oxidation Chemistry of Adenosine-3′, 5′-Cyclic Monophosphate at Pyrolytic Graphite Eletrode

The voltammetric oxidation of adenosine-3′,5′-cyclic monophosphate (3′,5′-CAMP) has been studied in the pH range 2.13–10.07 using pyrolytic graphite electrode (PGE). Voltammetric, coulometric, spectral studies, and product characterization indicate that the oxidation of 3′,5′-CAMP occurs in an EC reaction involving a 6H⁺, 6e process at pH 7.24. Electrooxidized products were seperated by semipreparative high performance liquid chromatography (HPLC) and were characterized by mp, ¹HNMR, FTIR, and GC-mass as allantoin cyclic ribose monophosphate and 3 dimers as the major products. A detailed interpretation of the redox mechanism of 3′,5′-CAMP also has been presented to account for the formation of various products.     

Purification and succinylation of cyclic GMP from large volume samples and radioimmunoassay of succinyl cyclic GMP.

Improved procedures for isolation of cyclic GMP and cyclic AMP and radioimmunoassay of cyclic GMP with succinylation are described. Procedures involved include modified chromatography on alumina and succinylation of cyclic GMP followed by purification of succinyl cyclic GMP on a Dowex AG 1×8 column. These procedures are convenient and applicable to any volume up to 50 ml of tissue extracts and especially for isotonic incubation mixtures. This assay system is sensitive to 6 femtomoles of cyclic GMP/tube. On radioimmunoassay, free and antibody bound [¹²⁵I]-labeled cyclic GMP are separated by Millipore filtration. Cyclic GMP levels in several tissue samples were determined in order to show the applicability of the procedures.     

Interaction between flavins and alcohols

The effect of alcohols on the spectral properties of riboflavin derivatives in non-polar solvent was studied by various spectroscopic methods in order to support the view point that alcohol may directly interact with the isoalloxazine moiety of FAD and enhance the catalytic activity of D-amino acid oxidase (DAAO). The most likely association complex between alcohol and riboflavin is 1 : 1 stoichiometric complex through the 3-N imino and the 2-C carbonyl groups of the isoalloxazine ring and the hydroxyl group of alcohols. It appears that methanol has a larger association constant than any other alcohols, and the association constant decreases with the increase in carbon number and with the steric requirement of the alkyl group of alcohols.     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Three single nucleotide polymorphisms leading to non-synonymous amino acid substitution in the human ribonuclease 2 and angiogenin genes exhibit markedly less genetic heterogeneity in six populations

Angiogenin and ribonuclease 2 (RNase 2) are members of the human RNase superfamily. Although three potential single nucleotide polymorphisms (SNPs) in these genes, which could give rise to an amino acid substitution in the protein, have been identified, relevant population data are not available, and accordingly they have not been applied to clinical-genetic analysis. For this purpose, a novel genotyping method for each SNP using the mismatched PCR-restriction fragment length polymorphism technique has been developed. Using this method, the genotype distribution of each SNP was investigated in six populations: Japanese (n = 167), Korean (n = 90), Mongolian (n = 92), Ovambos (n = 86), Turkish (n = 87), and German (n = 70). In all the populations, only one genotype was found in each SNP. Irrespective of differences in ethnic groups, the angiogenin and RNase 2 genes appear to exhibit markedly less genetic heterogeneity with regard to these SNPs.     

New flavonoids from the underground parts of Eriosema laurentii

Pharmacological, toxicological and phytochemical investigations of aerial parts of Eriosema laurentii De Wild. (Leguminosae), a plant used in traditional African medicine, showed a complex pattern of very rare and several new exceptional phenolic compounds. In continuation of the phytochemical work and based on the activity of an extract from the underground parts, the presented study showed the occurrence of a new pyranoisoflavone, named methyleriosemaone D, and a new highly prenylated (2S,3S)-6,8,3′-triprenyl-3,5,7,2′,4′-pentahydroxyflavanone in these parts of E. laurentii. In addition, the very rare pyranoisoflavone eriosemaone D was proven in this plant for the first time. All structures were elucidated unambiguously by extensive MS- and one and two-dimensional NMR-experiments. Detailed NMR spectra of eriosemaone D were included to rationalize the correction of some of the previously reported carbon signals in the ¹³C NMR spectrum. The estrogenic activity of the extract and isolated compounds was tested in an estrogen receptor α yeast transactivation assay.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

A New Bipyrrole and Some Phenolic Constituents in Prunes (Prunus domestica L.) and Their Oxygen Radical Absorbance Capacity (ORAC)

Isolation and structural elucidation of prune constituents were performed and total 10 compounds were determined by NMR and MS analyses. A novel compound was identified to be 2-(5-hydroxymethyl-2′,5′-dioxo-2′,3′,4′,5′-tetrahydro-1′H-1,3′-bipyrrole)carbaldehyde, and 7 phenolic compounds were isolated from prunes for the first time. In addition, antioxidant activity of them was evaluated on the basis of the oxygen radical absorbance capacity (ORAC).     

Regulation of ketone body production from (14C)palmitate in rat liver mitochondria: effects of cyclic nucleotides and unlabeled fatty acids.

N⁶′, O²′-dibutyryl adenosine 3′, 5′-cyclic monophosphoric acid, but not other cyclic nucleotides stimulates [¹⁴C]ketone body production from [¹⁴C]palmitate in isolated rat liver mitochondria. Butyrate alone, as well as unlabeled acetate, octanoate and palmitate had similar effects. This redistribution of the oxidative products of [¹⁴C]palmitate can best be explained by exceeding the capacity of the Krebs cycle and/or changes in the acetyl coenzyme A/coenzyme A ratio. In contrast to [¹⁴C]palmitate, [¹⁴C]octanoate oxidation to [¹⁴C]O₂ and [¹⁴C]ketone bodies was inhibited by the addition of unlabeled fatty acids. This suggests that an additional mechanism by which unlabeled fatty acids may stimulate [¹⁴C]ketone body production is by enhancing the carnitine-dependent transport of [¹⁴C]palmitate into mitochondria.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats

In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical sympathectomy and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats.Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP content of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but not phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue.Thus we have established the responsiveness of the cyclic GMP content of brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the α-adrenergic stimulation and in the regulation of proliferative processes in the tissue.     

Two new C-methylated flavonoids from Myrica gale

From the leaves of Myrica gale 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone has been isolated. The fruits yielded 2′-hydroxy-4′,6′-dimethoxy-3′-methyldihydrochalcone. The constitutions were deduced from spectroscopic data and confirmed by synthesis.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.