Microbial Asymmetric Reduction. Preparation of Optically Active Methyl trans-3-(4-Methoxyphenyl)Glycidates

As a result of screening of microorganisms, Mucor ambiguus IFO 6742 was found to reduce methyl 2-chloro-3-(4-methoxyphenyl)-3-oxopropionate (2) to give methyl (2S,3R)-2-chloro-3-hydroxy-3-(4-methoxyphenyl)propionate [(2S,3R)-3] in good yield with high enantioselectivity. The resulting (2S, 3R)-3 was converted into methyl (2S,3R)-3-(4-methoxyphenyl)glycidate [(2S,3R)-4] by treatment with sodium methoxide. On the other hand, its enantiomer, (2R,3S)-4 was obtained by the Mitsunobu esterification of (2S,3R)-3 and subsequent treatment with sodium methoxide. Also (2R,3S)-4 was obtained by the treatment of (2RS,3S)-3, which was obtained from 2 by Trichoderma viride OUT 4642, with sodium methoxide.     

Synthesis and anti-HIV evaluation of 2',3'-dideoxy imidazo- and nu-triazolo[4,5-d]pyridazine nucleosides

The syntheses of the 2'-deoxy and 2',3'-dideoxynucleosides of 2,8-diaza-3-deazainosine and the 2',3'-dideoxynucleoside of 2-aza-3-deazainosine were achieved and the pathways leading to these novel nucleosides are described. The preparation of the 2',3'-dideoxynucleoside (1) of 2-aza-3-deazainosine involved deoxygenation of the 2'-deoxy-3'-imidazolide intermediate with n-Bu3SnH and AlBN. The latter nucleoside was synthesized from the known 2'-deoxy derivative of 2-aza-3-deazainosine. The three-step synthesis of 1 from the 2'-deoxy analogue was accomplished in 40% overall yield. Rather than synthesize the corresponding 2',3'-dideoxynucleoside (2) of 2,8-diaza-3-deazainosine in the same manner, i.e. deoxygenation of the 2'-deoxynucleoside, a more cost-effective route was chosen. This pathway involved reductive cleavage of the 5'-protected, 2',3'-thiocarbonate derivative to furnish a mixture of the 2'- and 3'-deoxy isomers. This mixture was not separated, but was deoxygenated by the aforementioned imidazolide method. Using this methodology, 2 was prepared in 23% overall yield from 2,8-diaza-3-deazainosine. Nucleosides 1 and 2 were evaluated for antiretroviral activity and were found to be inactive.     

Substrate stereochemistry of the enoyl-CoA hydratase reaction.

1. A specimen of stereospecifically 2-tritiated 3-hydroxybutyric acid was prepared by hydroboration of ethyl crotonate. It was assumed that the hydroboration reaction took a syn course and hence that (2R,3S) plus (2S,3R)-3-hydroxy[2 minus 3H1]butyric acid was formed after oxidation and hydrolysis. 2. 3RS-3-Hydroxy[2 minus 3H2]butyric acid, symmetrically tritiated at C-2, was prepared by isotopic exchange of ethyl acetoacetate in tritiated water, followed by reduction and hydrolysis. 3. The 3R-enantiomers of the acids listed under paragraphs (1) and (2) were destroyed enzymically by use of 3R-specific 3-hydroxybutyrate dehydrogenase and the residual 3S-enantiomers were isolated. 4. The resulting specimens of 2R,3S-3-hydroxy[2 minus 3H1]butyric acid and 3S-3hydroxy[2 minus 3H2]-butyric acid were converted chemically to the acyl-CoA derivatives. These were incubated with enoyl-CoA hydratase. 5. In the presence of the enoyl-CoA hydratase symmetrically labelled 3S-3-hydroxy[2 minus 3H2]BUTYRYL-CoA lost nearly 50% of its tritium label; 2R,3S-3hydroxy [2-3-H1]butyryl-CoA lost about 78%. 6. It was concluded that the elimination of the elements of water from 3-hydroxybutyryl-CoA on the hydratase occurs stereospecifically with syn geometry.     

Effects of estradiol-17 beta and estriol on their binding sites in the rabbit uterus

1. Receptors for estradiol-17 beta (E2) and estriol (E3) were detected in the rabbit uterus. 2. Saturation analysis of estrogen binding sites in the cytosol showed that the dissociation constants of E2 and E3 for the high affinity binding sites were 1.8 +/- 0.5 nM and 2.3 +/- 0.3 nM, respectively, when dextran-coated charcoal was used to isolate free and bound ligands. 3. To eliminate non-specific (cross) bindings to their receptors, effects of unlabeled E2 and E3 on [3H]E3 and [3H]E2 bindings was examined. 4. [3H]E2 cytosol binding was observed to be specific for E2 and [3H]E3 cytosol binding was more specific for E3. 5. E2 priming to rabbits increased the binding sites for both E2 and E3, which was also more potent than E3 priming. 6. Moreover, the increase in E2 binding sites was greater than that in E3 binding sites. 7. These findings may suggest that there are separate binding sites for E2 and E3 in rabbit uterus and that synthesis of their binding sites is regulated by E2 but not E3.     

Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery

Improved non-viral vector systems are needed for efficient delivery of DNA to target cell nuclei in gene therapy. A series of linear polyamine poly(ethylene glycol) (PEG) constructs has been synthesised by reaction of appropriately Boc-protected thermine derivatives with omega-methoxyPEG oxiranylmethyl ethers. Constructs carrying 1-3 MeOPEG units and 0, 2 or 4 N-methyl groups have been prepared by this method. H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NHBoc was prepared efficiently by mono-trifluoroacetylation of thermine, attachment of Boc and removal of the trifluoroacetyl group in one pot. A similar process gave H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2. BocMeN(CH2)3NHMe was alkylated by 1,3-dibromopropane to give BocMeN(CH2)3NMe(CH2)3NMe(CH2)3NMeBoc. A cyanoethylation/reduction sequence extended H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2 to give H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2) 3NH2, which was converted to its mono- and di-MeOPEG550 derivatives. Deprotection gave the linear polyamine MeOPEG constructs. A branched triamine-poly(ethylene glycol) construct was prepared by acylation of (BocHN(CH2)3)2N(CH2)3NH2 with omega-methoxyPEG 550 chloroformate, followed by deprotection. A cyanoethylation/reduction/protection sequence from (H2N(CH2)3)2 N(CH2)3NHBoc gave a protected pentamine. Alkylation with Br(CH2)5CONH(CH2)2NHBoc, deprotection, acylation with MeOPEG chloroformate and deprotection gave a pentamine MeOPEG construct in which the MeOPEG is attached through a linker to the central amine. The linear hexamine construct carrying MeOPEG550 at only one terminus was the most effective DNA-interactive member of the two series in an ethidium displacement assay and was effective in delivering a reporter gene to RIF-1 tumours.     

表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定

为研制一种预防犬科动物狂犬病的新型疫苗,将含有狂犬病毒ERA株糖蛋白基因(Rabies glycoprotein,Rgp)表达盒的穿梭质粒pVAXΔE3Rgp中的Rgp表达盒克隆入犬2型腺病毒(Canine adenovirus type2,CAV2)骨架质粒pPoly2-CAV2中,获得重组质粒pPoly2-CAV2-ΔE3-Rgp,释放其基因组,转染MDCK细胞系,获得E3缺失区(Deletion of early protein3,ΔE3)含有Rgp表达盒的重组病毒CAV2-ΔE3-Rgp。该重组病毒能在MDCK细胞上产生典型的腺病毒细胞病变。通过酶切、PCR、基因测序,表明该重组病毒含有完整的Rgp表达盒。通过RT-PCR、Western blot等检测,表明该重组病毒能够表达Rgp抗原。用该重组病毒免疫犬,3次免疫后,可以诱导犬产生特异的抗CAV2HI抗体,其效价超过1∶256和抗狂犬病病毒(Rabies virus,RV)中和抗体,其效价超过0.50IU/mL。试验结果表明,获得的重组病毒免疫犬后,能够产生抗狂犬病毒和腺病毒的高效价保护性抗体,是一种有潜力的犬科动物狂犬病毒腺病毒二联疫苗候选株。     

Synthesis and separation of diastereomers of uridine 2',3'-cyclic boranophosphate

The first boron-containing 2',3'-cyclic phosphate-modified analogue, uridine 2',3'-cyclic boranophosphate (2',3'-cyclic-UMPB), was synthesized. 5'-O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2',3'-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2',3'-cyclic-UMPB in high yield. The two diastereomers of 2',3'-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2',3'-cyclic phosphorothioate (2',3'-cyclic-UMPS) via H-phosphonate was also described.     

Specific localization of 2',3'-cyclic nucleotide 3'-phosphohydrolase, (Ca2+/Mg2+)-ATPase, and acetylcholinesterase in human erythrocyte membrane

A procedure was developed for the detection of 2',3'-cyclic nucleotide 3'-phosphohydrolase in myelin. This assay was sufficiently to detect the low levels of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human erythrocytes. The 2',3'-cyclic nucleotide 3'-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghosts and resealed ghosts were assayed for 2',3'-cyclic nucleotide 3'-phosphohydrolase (Ca2+/Mg2+)-ATPase, and acetylcholinesterase activity, and the 2',3'-cyclic nucleotide 3'-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATP, an established inner membrane marker.     

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.     

The infusion of trieicosapentaenoyl-glycerol into humans and the in vivo formation of prostaglandin I3 and thromboxane A3

Thirty ml of an emulsion containing 3 g of trieicosapentaenoyl-glycerol (90% pure, containing 5% arachidonic acid (AA)) was infused intravenously in 2 male healthy volunteers. Urine samples were collected for 24 h before and 48 h after the infusion in 5 periods. Urinary metabolites of prostaglandin (PG) I2/3 and thromboxane (TX) A2/3 (PGI2/3-M and TXB2/3-M, respectively) were extracted from the urinary samples and measured by GC-MS. Excretion of PGI3-M was markedly enhanced right after the infusion. Because PGI3 was produced without involvement of intestinal absorption of eicosapentaenoic acid (EPA), enhanced PGI3 formation was strongly suggested to take place in the vasculature. From the marked increment in TXB2/3-M after the infusion it was calculated that conversion rate of EPA to TXA3 was 8% of that of AA to TXA2 in this in vivo condition.     

Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor.

The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

l-methionine-γ-lyase in Citrobacter intermedius cells: Stereochemical requirements with respect to the thiol structure

The specificity of -methionine-γ-lyase with respect to the stereochemical structure of the thiol substrate in the γ-substitution reaction has been demonstrated. Cells of Citrobacter intermedius containing -methionine-γ-lyase catalyze the exchange reactions between -methionine and 2-propylthiol or 2-butylthiol which leads to the formation of -2-propylhomocysteine and -2-butylhomocysteine, respectively. The yields of these products are comparable to the yield of -butylhomocysteine in the reaction of normal butylthiol with -methionine, thus 2-propylthiol and 2-butylthiol are effective substrates of -methionine-γ-lyase. On the other hand in the reaction of 3-pentylthiol, only traces of the expected product, -3-pentylhomocysteine, were formed and in the case of 2-methyl-2-butylthiol, the expected product of γ-substitution, -2-methyl-2-butylhomocysteine, was not formed at all. In the reaction with racemic 2-butylthiol, only one diastereomer of -2-butylhomocysteine was obtained. The unreacted 2-butylthiol isolated after the reaction catalyzed by partially purified preparation of -methionine-γ-lyase was enriched with (R)-enantiomer which indicated the preferential reaction of the (S)-enantiomer.     

Ionic mechanism of Na+-HCO3- cotransport in rabbit renal basolateral membrane vesicles

The exit of HCO3- across the basolateral membrane of the proximal tubule cell occurs via the electrogenic cotransport of 3 eq of base per Na+. We have used basolateral membrane vesicles isolated from rabbit renal cortex to identify the ionic species transported via this pathway. Media of varying pH and pCO2 were employed to evaluate the independent effects of HCO3- and CO3(2-) on 22Na transport. Na+ uptake was stimulated when [CO3(2-)] was increased at constant [HCO3-], indicating the existence of a transport site for CO3(2-). In the presence of HCO3-, Na+ influx was stimulated more than 3-fold by an inward SO3(2-) gradient. SO3(2-)-stimulated Na+ influx was stilbene-sensitive, confirming that it occurs via the Na+-HCO3- cotransport system. Na+-SO3(2-) cotransport was demonstrated and found to have a 1:1 stoichiometry. Increasing [CO3(2-)] at constant [HCO3-] reduced the stimulation of Na+ influx by SO3(2-), suggesting competition between SO3(2-) and CO3(2-) at a common divalent anion site. Additional divalent anions that were tested, such as SO4(2-), oxalate2-, and HPO4(2-), did not interact at this site. SO3(2-) stimulation of Na+ influx was absolutely HCO3-(-)dependent and was increased as a function of [HCO3-], indicating the presence of a separate HCO3- site. Lastly, we tested whether Na+ interacts via ion pair formation with CO3(2-) or binds to a distinct site. Na+, which has lower affinity than Li+ for ion pair formation with CO3(2-), was found to have greater than 5-fold higher affinity than Li+ for the Na+-HCO3- cotransport system. Moreover, when its inhibition was studied as a function of [Na+], harmaline was found to be a competitive inhibitor of Na+ influx, indicating the existence of a distinct cation site. Our data are compatible with a model in which base transport across the basolateral membrane of the proximal tubule cell takes place via 1:1:1 cotransport of CO3(2-), HCO3-, and Na+ on distinct sites.     

Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage

Incubation of the pyrimidine [3'-3H]UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP. During this reaction 3H2O was also released. The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced. At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized. Similar experiments in which the purine [3'-3H]ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4. Furthermore, 3H2O was also released as a function of the extent of the reaction. At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released. Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with [3'-3H]ADP or [3'-3H]UDP indicated that in neither case was 3H released. Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond. Incubation of [3'-3H]dADP or [3'-3H]dUDP with ribonucleotide reductase produced no measurable release of 3H. These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate. The fate of the 3'-H of the substrate was also determined. Incubation of [3'-2H]UDP with ribonucleotide reductase resulted in the production of [3'-2H]dUDP.     

Effect of substitution inert metal complexes on calcineurin.

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.     

Visible light decomposition of ammonia to N2 with Ru(bpy)3(2+) sensitizer.

Visible light decomposition of aqueous NH3 to N2 was investigated using a photocatalyst aqueous solution based on molecular photoelectron relay systems of either sensitizer (tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+))/potassium peroxodisulfate(K(2)S(2)O(8)) or Ru(bpy)3(2+)/methylviologen dichloride(MV2+)/O2, capable of using visible light instead of UV-driven semiconductors such as TiO2. It was confirmed by using an in situ visible absorption spectral change under irradiation that the Ru(II) complex is oxidized to the Ru(III) complex by K(2)S(2)O(8), and that the Ru(III) complex formed is stable without NH3, while the added NH3 was oxidized by the Ru(III) complex to produce the Ru(II) complex. In the presence of 1 mM NH3 aqueous solution, the Ru(III) complex was the predominant species under the photostationary state, but in the presence of 100 mM NH3, Ru(II) predominated. Gas-chromatographic analysis of the gaseous phase in the presence of 8.1 M NH3 showed that the photochemical oxidation of ammonia yielded N2. It was also demonstrated by using the in situ visible absorption spectrum under irradiation of the NH3 (1 M)/Ru(bpy)3(2+) (0.1 mM)/MV2+ (10 mM) system under Ar that MV+* is accumulated, showing that NH3 works as an electron donor for MV+* accumulation with simultaneous formation of the oxidized product of ammonia ((NH3)ox) without producing N2. It was suggested that the reduced product (MV+*) and the oxidized product ((NH3)ox) are in a kind of dynamic equilibrium prohibiting further oxidation of (NH3)ox by Ru(bpy)3(3+) to N2. In the O2 atmosphere, the oxidation of MV+* to MV2+ takes place to accumulate Ru(III) complex, so that (NH3)ox was further oxidized to N2. The high activity of IrO2 as a cocatalyst in this system was demonstrated.     

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.