The 3' 5' exonucleases

Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.     

The targets of CAPRI rounds 3-5

Ten protein-protein complexes have been offered by X-ray crystallographers as targets for structure prediction in Rounds 3-5 of the CAPRI experiment. They illustrate molecular recognition in several domains of biology: enzyme regulation, antigen-antibody recognition, signal transduction, and oligomer assembly. The targets presented various degrees of difficulty to the predictors, depending on their status (bound when components were taken from the complex, unbound when coming from independent structures of the free proteins), the amplitude of conformation changes, and the amount of biological information available. Predictors produced high-quality models of 6 of the targets, good models of 3 others, and failed only in 1 case, where the conformation change was particularly large. This result demonstrates significant progress relative to earlier rounds of CAPRI.     

The effective synthesis of uridylyl/3''-5''/5-methylcytidylyl/3''-5''/guanosine.

The triester method was adapted to the synthesis of uridylyl/3'-5'/5-methylcytidylyl/3'-5'/guanosine. As the protecting groups 4-methoxy-5,6-dihydro-2H-pyran for 2'-OH and 5'-OH groups of uridine and 2'-OH group of 5-methylcytidine, methoxymethylidene for I:3'-cis-diol system of guanosine, and benzoyl for the amino groups of 5-methylcytidine and guanosine were used. The obtained product was characterised by UV, electrophoresis, chromatography, an enzymatic digestion and alkaline hydrolysis.     

The reduction of 5 alpha-cholestan-3-one and 5 beta-cholestan-3-one by some boranes and hydroborates.

This paper describes the high yield synthesis of 5 alpha-cholestan-3 alpha-ol and 5 beta-cholestan-3 beta-ol by the reduction of 5 alpha- and 5 beta-cholestan-3-one using using potassium and lithium-tri-sec-butyl-hydroborates as reducing reagents. Attempts to obtain the same alcohols using other bulky boranes resulted in the equatorial products.     

The penetration of 3:5-dinitro-o-cresol into leaves

A study of the penetration of 3:5-dinitro- 0 -cresol from spray solutions or suspensions into leaves of Sinapis arvensis L. shows that entry through the stomata is unimportant. Lethal amounts may enter from aqueous solution by simple diffusion of the undissociated phenol through the epidermis, which behaves as a homogeneous lipide membrane. The effect of the structure of the epidermal surface on the rate of diffusion is discussed. Lethal amounts may also enter Sinapis from a dry surface deposit by gaseous diffusion through the stomata or by diffusive penetration of the epidermis following sublimation. Movement of the substance within a plant is slight and appears to be mainly by diffusion. The selective action of 3:5-dinitro- 0 -cresol as a weed-killer is discussed in the light of these results.     

The 5′-Purine-Pyrimidine-3′ Stacks are More Stabilizing in a Self-3′-Pyrinudine-Purine-5′ complementary DNA Duplex than the 5′-Purine-Purine-3′ 3′-Pyrimidine-Pyrimidine-5′ Stack

Abstract

First experimental evidence is herein reported supporting the earlier quantum chemical calculations that 5′-Punne-pyrimiidine-3′ 3′ -Pyrimidine-Punne-5 stack is more stable than 5′-Pyrimiidine-Punne-3′ 3′-Punne-Pyrimidine-5′.     

The synthesis of protected 5''-amino-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; applications of 5''-amino-oligodeoxyribonucleotides.

Synthetic routes to the four appropriately protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) have been developed. The structures of all intermediates were confirmed by 13C n.m.r. spectroscopy. These building blocks have been used to prepare 5'-amino-oligodeoxyribonucleotides, which can be coupled to a wide variety of compounds, in particular metal cluster derivatives, but also fluorophores and biotin derivatives, thus generating a variety of very useful probes. Brief mention is made of a tetrairidium cluster derivative of 5'-amino-d[CCGATATCGG], which has been cocrystallised with EcoRV, and will be used for electron microscopy studies.     

The synthesis of protected 5''-mercapto-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; uses of 5''-mercapto-oligodeoxyribonucleotides.

The syntheses of the four novel, base protected 5'-(S-triphenylmethyl)mercapto-2',5'-dideoxyribonucleoside-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) are described. These compounds have been used to prepare 5'-(S-triphenylmethyl) mercapto-oligodeoxyribonucleotides, which are readily purified by reversed phase h.p.l.c., owing to the highly lipophilic trityl group. After cleavage of the S-trityl group by silver or mercuric ions, the free thiol moiety can be coupled to a wide variety of reagents, generating very useful probes. Fluorescent labelled 5'-mercapto-oligodeoxyribonucleotides are being used for automated DNA sequencing without radioactivity, and heavy metal labelled 5'-mercapto-oligonucleotides will be used in X-ray crystallography.     

A regiospecific synthesis of branched tetranucleotides: U3'p5'A2'p5'G 3'p5'U and U3'p5'A2'p5'G3'p5'C

An efficient strategy for the synthesis of branched nucleotides 14 and 15 has been developed using key intermediates 6 and 10.     

The 5-HT3B subunit confers spontaneous channel opening and altered ligand properties of the 5-HT3 receptor

Current receptor theory suggests that there is an equilibrium between the inactive (R) and active (R*) conformations of ligand-gated ion channels and G protein-coupled receptors. The actions of ligands in both receptor types could be appropriately explained by this two-state model. Ligands such as agonists and antagonists affect receptor function by stabilizing one or both conformations. The 5-HT3 receptor is a member of the Cys-loop ligand-gated ion channel superfamily participating in synaptic transmission. Here we show that co-expression of the 5-HT3A and 5-HT3B receptor subunits in the human embryonic kidney (HEK) 293 cells results in a receptor that displays a low level of constitutive (or agonist-independent) activity. Furthermore, we also demonstrate that the properties of ligands can be modified by receptor composition. Whereas the 5-hydroxytryptamine (5-HT) analog 5-methoxyindole is a partial agonist at the 5-HT3A receptor, it becomes a "protean agonist" (functioning as an agonist and an inverse agonist at the same receptor) at the 5-HT3AB receptor (after the Greek god Proteus, who was able to change his shape and appearance at will). In addition, the 5-HT analog 5-hydroxyindole is a positive allosteric modulator for the liganded active (AR*) conformation of the 5-HT3A and 5-HT3AB receptors and a negative allosteric modulator for the spontaneously active (R*) conformation of the 5-HT3AB receptor, suggesting that the spontaneously active (R*) and liganded active (AR*) conformations are differentially modulated by 5-hydroxyindole. Thus, the incorporation of the 5-HT3B subunit leads to spontaneous channel opening and altered ligand properties.     

The Central 5-HT3 Receptor in CNS Disorders

Among the characterized 5-HT receptors of the central nervous system, the type 3 receptor subtype (5-HT₃R) is the only one known to be a ligand-gated ion channel. Its early pharmacological characterization and mapping by radioligand binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigro-striatal pathway and reduce alcohol consumption in experimental animals while antagonists of this receptor have been reported to treat anxiety disorders. Following the cloning of this receptor in 1991, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis. Here we summarize our recent efforts showing that 5-HT₃R-expressing neurons are mainly GABA containing cells in the rat neocortex, olfactory cortex, hippocampus, and amygdala which also often contain chole-cystokinin (CCK) immunoreactivity. These results provide a means to unify some of the initial pharmacological observations.     

The 3'-amido and 5'-amido analogues of adenosine 3':5'-monophosphate; interaction with cAMP-specific proteins.

The sensitivity for recognition of adenosine 3:5'-monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six-membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3' and 5'-amido analogues (I, II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart. Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reeuced relative to cAMP, the 3'-position being more sensitive towards the modification than the 5'-position. The coordinate noncyclic derivatives, 3'-deoxy-3'-amino-5'-AMP (IV) and 5'-deoxy-5'-amino-3'-amp (iii), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5'-deoxy-5'-amino-3'-AMP (III), while the 3'-deoxy-3'-amino-5'-AMP (IV) is practically inactive. A possible reason for this is that the noncylic 5'-analogue (III) may be able to assume a cyclic structure maintained by internal salt formation. The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5'-analogue (I) than the 3'-analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I. The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X-ray work).     

Anti-viral activity of 3-deazaadenosine and 5'-deoxy-5'-isobutylthio-3-deazaadenosine (3-deaza-SIBA)

3-Deazaadenosine and 5′-deoxy-5′-isobutylthio-3-deazaadenosine (3-deaza-SIBA) inhibits replication of both herpes simplex type 1 virus and the RNA type C virus, HL-23. Oncogenic transformation caused by SV40 and HL-23 are also blocked by either compound. Both compounds exhibit relatively low cytotoxicity at the anti-viral concentrations.     

The metabolism in man of (3H)-5alpha-16-androsten-3alpha-ol and of (3H)-5alpha-16-androsten-3-one

The in vitro metabolism in man of two 16-androstene steroids, 5alpha-16-androsten-3-one and 5alpha-16-androsten-3alpha-ol, has been studied using 3H-labelled tracers. 4 healthy subjects (2 of each sex) were chosen, and each labelled steroid was administered, by a single injection, to 1 man and 1 woman. Disappearance of (3H)-3alpha-androstenol in the subjects receiving this compound followed a curve which indicated a two-pool distribution in both cases; metabolic clearance rates for these subjects were found to be 3,790 1/24 h in the man and 3,120 1/24 h in the woman. Blood production rates calculated for the 3alpha-androstenol-treated subjects were 875 microgram/24 h in the man and 1,780 microgram/24 h in the woman. Recovery of 3H in the urine of all 4 subjects was very low, between 28 and 42%. Conversion of the injected precursors to urinary 3alpha-androstenol was 13.5 and 12.7% in the 2 men and 6.1 and 5.9% in the 2 women. The male subjects were found to have a lower 24-hour urinary 3alpha-androstenol output (570 and 387 microgram/24 h) than the average for men of their age. The urinary 3alpha-androstenol output in the women was 225 and 276 microgram/24 h, and was within the normal range for women. The urine production rates of 3alpha-androstenol were 2,470 and 4,090 microgram/24 h in the male and female subjects, respectively; the difference between the blood and urine production rates of this compound are thought to indicate the direct secretion of conjugates. Urine production rates of 5alpha-androstenone (measured as 3alpha-androstenol) were 2,370 and 4,340 microgram/24 h in the male and female subjects, respectively.     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.