Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

5. GENERAL NOTES

Olive Thrushes Turdus olivaceus olivaceus in Grahamstown, South Africa, were present in their territories throughout the year. Mist-net captures showed no seasonal fluctuation in the population density of adults. The breeding season had a main peak from August to November and a minor peak in April. Song output was greatest at the beginning of the breeding periods, when territorial boundaries were most keenly contested. There was a positive correlation between seasonal variation in breeding intensity and song output. The adult survival rate was estimated at 80%, and 56% of the fledglings were alive in their natal territories at the age of independence. In this study area, only females built nests and brooded nestlings. Only males sang. Both males and females defended their territories.     

Affinity labelling of 5''-nucleotidases with 5''-p-fluorosulphonylbenzoyladenosine.

5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.     

Pusillimonas sp. 5HP degrading 5-hydroxypicolinic acid

A bacterial strain 5HP capable of degrading and utilizing 5-hydroxypicolinic acid as the sole source of carbon and energy was isolated from soil. In addition, the isolate 5HP could also utilize 3-hydroxypyridine and 3-cyanopyridine as well as nicotinic, benzoic and p-hydroxybenzoic acids for growth in the basic salt media. On the basis of 16S rRNA gene sequence analysis, the isolate 5HP was shown to belong to the genus Pusillimonas. Both the bioconversion analysis using resting cells and the enzymatic assay showed that the degradation of 5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic acid was inducible and proceeded via formation of the same metabolite, 2,5-dihydroxypyridine. The activity of a novel enzyme, 5-hydroxypicolinate 2-monooxygenase, was detected in the cell-free extracts prepared from 5-hydroxypicolinate-grown cells. The enzyme was partially purified and was shown to catalyze the oxidative decarboxylation of 5-hydroxypicolinate to 2,5-dihydroxypyridine. The activity of 5-hydroxypicolinate 2-monooxygenase was dependent on O₂, NADH and FAD.     

5''-Halogeno-2'',3''-cyclic sulphite isomers in the preparation of 5''-halogeno nucleosides. Synthesis of 5''-deoxyuridine and 5''-deoxy-5-fluorouridine.

When uridine (Ia) is reacted with thionyl chloride in hexamethylphosphoric triamide a mixture of isomeric 5'-chloro-2',3'-sulphites is formed, which can be separated to individual epimers IIa and IIIa, in 45% and 15% yields, respectively. Analogously, crystalline epimers IIb (37%) and IIIb (17%) can be obtained from 5-fluorouridine (Ib). Both isomers IIa, IIIa (or IIb, IIIb) afford a single 5'-chloro derivative IVa (or IVb, respectively) if treated with 0.1N sodium methoxide. From the mixture of sulphites IIa and IIIa (or IIb and IIIb) crystalline 5'-chlorouridine IVa is formed in 84.5% yield, calculated per starting uridine Ia (or crystalline 5'-chloro-5-fluorouridine IVb, 85.5% per starting 5-fluorouridine Ib, respectively). On reduction of 5'-chlorouridine IVa with tributyltin hydride 5'-deoxyuridine (Va) is formed in 79% yield. During the reduction of 5'-chloro-5-fluoro derivative IVb to 5'-deoxy-5-fluorouridine (Vb, 57%) a partial reductive elimination of 5-fluorine takes place under formation of 5'-deoxyuridine (Va, 9%).     

Raman ph profiles for nucleic acid constituents. II. 5'-AMP and 5'-GMP ribonucleotides.

Raman spectra of aqueous solutions of 5'-AMP and 5'-GMP have been recorded as a function of pH. Band intensities and frequencies have been monitored to determine bands which may be considered as diagnostic for the concentration and the pK of the solution species. Quantitative band intensity measurements indicate only a selected number of bands can be considered as diagnostic of the base or the secondary phosphate proton dissociation. The utility of the pH profiles derived from specific band intensity variations for 5'-AMP is discussed in terms of the effect of acidification on solution characteristics of polyadenylic acid.     

Preparation of Oligonucleotides Containing 5-Bromouracil and 5-Methylcytidine.

Abstract

A previously described side reaction on 5-bromouracil during standard oligonucleotide deprotection conditions has been studied in detail. The side product, 5-amino-2′-deoxyuridine, is isolated and characterized. The use of several 5-methylcytidine protected derivatives for the preparation of oligonucleotides containing 5-bromouracil and 5-methylcytidine free of 5-amino-2′-deoxyuridine is discussed.     

Partie 5. Pathologies tumorales

Bone scintigraphy is a nuclear imaging scan using a radiopharmaceutical composed of a bisphosphonate coupled to a radionuclide (technetium 99 m). Radiopharmaceutical uptake is particularly important at the level of the bone structures having a strong osteoblastic activity. These uptakes can be due to a benign pathology (fracture, loosening of prosthesis, rheumatic pathologies, etc.) or to a malignant pathology (primary or secondary bone lesion). The high sensitivity of bone scintigraphy makes it particularly interesting at the initial stage of the pathology, especially when X-rays are normal. In addition, its specificity has clearly improved in recent years with the increasingly use of tomoscintigraphy coupled with X-ray scanning (SPECT/CT). We describe the operating principle of bone scintigraphy, normal uptakes with its variants as well as pathological uptake features in traumatic, rheumatic, prosthetic or cancerous pathologies.     

5. NOTES FROM GRAAFF-REINET

Clark, A. 1978. Some aspects of the behaviour of whistling ducks in South Africa. Ostrich 49:31-39.

The behaviour of the Whitefaced Whistling Duck Dendrocygna viduata and the Fulvous Whistling Duck D. bicolor wasstudied between 1971 and 1974 on the Witwatersrand, Transvaal. The paper describes and compares behaviour associated with feeding, voice, flight, agonistic situations and copulation in the two species.     

Restriction insensitivity in bacteriophage T5. II. Lack of EcoRI modification in T5+ and T5ris mutants.

Neither bacteriophage T5+ nor its EcoRI-sensitive ris mutants became modified during growth on an EcoRI-modifying host. For this reason, the rare ris plaques able to grow on the EcoRI-modifying host were always due to revertant phage rather than to modified ris mutants. The ris mutations resulted in the creation of new EcoRI cleavage sites in the terminally repetitious first-step transfer DNA, and analysis of T5 ris revertants showed loss of these sites and restoration of the wild-type restriction pattern. Natural EcoRI sites present in the second-step transfer DNA were never lost in T5ris revertants, indicating that these are irrelevant to in vivo restriction and are protected during growth on the restricting host.