Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).     

Convenient synthesis of 2'-deoxy-2-fluoroadenosine from 2-fluoroadenine

A convenient synthesis of 2'-deoxy-2-fluoroadenosine from commercially available 2-fluoroadenine is described. The coupling reaction of silylated 2-fluoroadenine with phenyl 3,5-bis[O-(t-butyldimethylsilyl)]-2-deoxy-1-thio-D-erythro-pentofuranoside gave the corresponding 2-fluoro-2'-deoxyadenosine derivative (alpha/beta = 1:1) in good yield. The alpha- and beta-anomers were separated by chromatography, and then desilylated to give compounds 1a and 1b.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone.     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Downregulation of ClC-2 by JAK2

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl(-) channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active (V617F)JAK2 or inactive (K882E)JAK2 and the Cl(-) channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Exposure of the oocytes expressing ClC-2 together with (V617F)JAK2 to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and (V617F)JAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (V617F)JAK2 and oocytes expressing ClC-2 alone, indicating that (V617F)JAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl(-) exit, an effect which may impact on cell volume regulation.     

New Aspect of H2O2 Signaling

Plant defense is based on a complex response triggered by unfavorable external impacts. The redox state of the cells and its temporal alteration, the oxidative burst, is an important regulatory element of this defense response. Data collected during the last years have caused us to change the previous, strongly simplified theory on signaling which had been based on a speculative, rather sequential mechanism. In the framework of signal transduction, H₂O₂ signaling pathway(s) is/are only a special part of signal transduction but interacting with other pathways it/they influence the whole transducting system in several points. Our results show that in complexity and in basic regulatory mechanisms (transients, oscillation, tuning, signaling pattern) H₂O₂ signaling is comparable with other pathways, of which we have more detailed cognition, and our present knowledge makes developing a new theory on this aspect necessary.Key Words: oxidative burst, elicitors, hydrogen peroxide, location, timing, long term monitoring, signal transduction     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Triplex forming ability of oligonucleotides containing 2'-O-methyl-2-thiouridine or 2-thiothymidine

The triplex forming ability of oligonucleotides containing 2'-O-methyl-2-thiouridine (s2Um) and 2-thiothymidine (s2T) was studied. The UV melting experiments revealed that triplex forming oligonucleotides (TFOs) containing both s2Um or s2T stabilized significantly parallel triplexes. The main reason for stabilization of triplexes was due to the stacking effect of the 2-thiocarbonyl group. Moreover, it turned out that these modified TFOs had a high selectivity in recognition of a matched Hoogsteen base from a mismatched one.     

Photooxidation of oligodeoxynucleotides by UO2(2+).

Photooxidation and consequent cleavage of oligodeoxynucleotides by the uranyl ion is greatly enhanced in the presence of a terminal 5'- or 3'-phosphate group. This enhanced cleavage is confined to nearby phosphodiester bonds in the case of a 5'-phosphate, but is less localized in the case of a 3'-phosphate. Several attempts to use a complementary oligodeoxynucleotide to direct a uranyl "warhead" against an oligodeoxynucleotide target were unsuccessful. Our results are most easily explained if we suppose that uranyl ions form coordination complexes with terminal phosphates and that, on photoexcitation, coordinated uranyl ions extract a hydrogen atom from the CH bond of a nearby deoxyribose residue.     

Computational determination of binding modes of 2-acetoxyphenylhept-2-ynyl sulfide to cyclooxygenase-2

Abstract

2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a potent covalent inhibitor with cyclooxygenase-2 (COX-2) selectivity. However, no crystal structure for APHS?COX-2 complex has been reported. In this work, we have extensively studied the binding modes and interactions between APHS and COX-2. Molecular docking followed by MD simulations identified a stable and reactive binding mode, of which the calculated binding free energy was in good agreement with the experimental reports. Furthermore, binding modes of six analogs of APHS were also analyzed to study the effects on binding affinity of the triple bond, heteroatom and length of alkyl chain. The findings help to understand the action mechanisms of APHS and explain why it is more potent than the analogs, which might be useful in the design of new compounds with higher inhibitory activity to COX-2.

Communicated by Ramaswamy H. Sarma     

Use of ERT2-iCre-ERT2 for conditional transgenesis

We examined the use of ERT2-iCre-ERT2 (Cre2ERT2), a tamoxifen-regulated form of Cre that has been described to have a background activity lower than that of other tamoxifen-regulated Cre constructs, for establishing performant conditional deleter mouse lines. Cre2ERT2 was inserted by homologous recombination into the Rosa26 locus. These mice were mated with R26R Cre-reporter mice. No recombination could be observed in the progenies in the absence of tamoxifen treatment. Tamoxifen treatment at E13-14 led to a high level, albeit variable, recombination in most of the tissues examined: liver, heart, kidney, brain, lung etc. Treatment of adult animals also induced recombination in these tissues, although at a lower level. Northern blot and qPCR studies suggested that these differences are not linked to significant variations of the level of expression of Cre2ERT2. Thus, Cre2ERT2 appears to be a good alternative to existing modulatable Cre systems, displaying a lack of background activity and a high-level inducibility in vivo.     

2H magnetic relaxation of 2H2O absorbed by wool.

D2O absorbed by intact wool fibers was studied by solid-state 2H nuclear magnetic resonance (NMR) spectroscopy. In wool fibers swollen in D2O, the deuteron transverse magnetization and the spin-locked magnetization revealed a non-exponential decay. At least two NMR phases with different sets of the NMR relaxation parameters, T(1rho) (2H) and T2 2H, have been detected that may be a manifestation of two different morphological phases of the cortex of the fiber.     

One-pot enzymatic production of 2-acetamido-2-deoxy-D-galactose (GalNAc) from 2-acetamido-2-deoxy-D-glucose (GlcNAc)

2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.