Diastereomerically pure nucleoside-5'-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s--substrates for synthesis of P-chiral derivatives of nucleoside-5'-O-phosphorothioates

A method for stereocontrolled chemical synthesis of P-substituted nucleoside 5'-O-phosphorothioates has been elaborated. Selected 3'-O-acylated deoxyribonucleoside- and 2',3'-O,O-diacylated ribonucleoside-5'-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s were chromatographically separated into P-diastereomers. Their reaction with anions of phosphorus-containing acids was highly stereoselective (≥90%) and furnished corresponding P-chiral α-thiodiphosphates and their phosphonate analogs with satisfactory yield.     

Synthesis and purification of fluorinated benzimidazole and benzene nucleoside-5'-triphosphates

The expression "universal base" is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g.,' their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.     

Patterns of mineralization in vitro

Summary Various patterns of mineralization are found in the organism during fetal and postnatal development. Different findings and theories have been published in the literature with regard to the mechanisms of mineralization, many of which are controversely discussed. In the present study the different patterns of mineralization observed in the organoid culture system of fetal rat calvarial cells were investigated by electron microscopy. In organoid culture, calvarial cells grow and differentiate at high density, and deposition of osteoid and mineralization of the matrix occur to a very high extent. Different types of mineralization could be observed more or less simultaneously. It was found that hydroxyapatite crystals were formed at collagen fibrils as well as in the interfibrillar space. Mineralization was frequently seen in necrotic cells and cellular remnants as well as in extra-and intracellular vesicles. Addition of bone or dentin matrices or the artificial hydroxyapatite Interpore 200 to the cells caused an increased mineralization in the vicinity and on the surface of the matrices with and without participation of collagen. On previously formed mineralized nodules, an apposition of mineralizing material appeared due to matrix secretion by osteoblasts. It is concluded that initiation of mineralization occurs-at least in vitro-at every nucleation point under appropriate conditions. These mineralization foci enlarge by further apposition as well as by cellular secretion of a mineralizing matrix. Furthermore, cell necroses may liberate mineralizable vesicles. All these patterns of mineralization are the result of different activities of one cell type.     

Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.     

Effects of chondroitin sulphates on mineralization in vitro

1. Soluble sodium chondroitin sulphate, from bovine ribs or puppy epiphyseal plate, at a concentration 80mm in terms of glucuronate, decreased the amounts of calcium and phosphate precipitated from a solution 6.9mm in phosphate and 6.9 or 13.8mm in calcium, buffered in the pH range 6.6-8.2 with 20-25mm-collidine and 5-20mn-hydrochloric acid when incubated for 2hr. at 30 degrees , and for 0.25-24hr. when buffered at pH7.0. 2. An insoluble fraction of puppy epiphyseal plate, containing chondroitin sulphate and collagen, was found to have the same effect at lower concentrations of chondroitin sulphate and a higher calcium/chondroitin sulphate glucuronate ratio, but the formation of calcium phosphate from calcium bound to this material appeared to proceed more rapidly than from calcium in solution, when both were present in the same system. 3. The implications of these results are discussed in relation to the calcium/chondroitin sulphate glucuronate ratios found in different parts of non-calcifying and calcifying cartilage in vivo and to the calcium and phosphate gradients between blood and calcified tissue.     

Indirect inactivation of rat brain cytosolic diacylglycerol kinase by nucleoside-5'-monophosphate

Cytosolic diacylglycerol kinase was inhibited drastically by nucleoside monophosphate. The inhibition was relatively specific for adenosine-5'-monophosphate (5'-AMP), although uridine-5'-monophosphate was also effective. The effect of 5'-AMP on diacylglycerol kinase appeared to be indirect since the degree of inhibition lessened with the dilution of the cytosol and the more purified enzyme failed to respond to 5'-AMP. A 5'-AMP-dependent mediator is proposed to be involved in the inactivation of diacylglycerol kinase.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Decorin modulates matrix mineralization in vitro

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.     

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines

cAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-TMP, and 5′-UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′-nucleotides suggest multi-nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.     

DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen

Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound.     

The specific, diverse effects of purine and pyrimidine nucleoside-5'-di(tri)-3'-diphosphates on the eucaryote translation system in vitro.

All the eight 5'-di(tri)-3'-diphosphates of four common ribonucleosides were prepared by the enzymic pyrophosphoryl transfer catalysed by Streptomyces adephospholyticus ATP:nucleotide pyrophosphokinase (E.C.2.7.6.4) form dATP to the respective 5'-phosphates, and their effects on the translation of mRNAs by a wheat germ system in vitro were studied. (p) ppPupp decreased the total 14C-leucine incorporation directed by a rat liver mRNA whereas (p) ppPypp did not. With a silkworm pupa ovary mRNA, distinctly reverase results were obtained. Gel electrophoretic profiles of the translation products disclosed the mRNA species-specific stimulatory or inhibitory effects for each of the polyphosphates tested.     

Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.