Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

The mode of toxic action of the pesticide gliftor: the metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrate

The biochemical toxicology of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with coenzyme A, ATP, oxaloacetate, and Mg2+ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg(-1) body weight) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 hour lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD+-dependent oxidation is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg(-1) body weight) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg(-1) body weight) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg(-1) body weight) to rats 2 hours prior to 1,3-difluoroacetone (100 mg kg(-1) body weight) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD+-dependent oxidation responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.     

1,3-Butadiene oxidation by human myeloperoxidase. Role of chloride ion in catalysis of divergent pathways.

1,3-Butadiene was oxidized by human myeloperoxidase in the absence of KCl to yield butadiene monoxide (BM) and crotonaldehyde (CA), but at KCl concentrations higher than 50 mM, 1-chloro-2-hydroxy-3-butene (CHB) was the major metabolite detected; metabolite formation was dependent on incubation time, pH, KCl, 1,3-butadiene, and H2O2 concentrations. The data are best explained by 1,3-butadiene being oxidized by myeloperoxidase by two different mechanisms. First, oxygen transfer from the hemoprotein would occur to either C-1 or C-4 of 1,3-butadiene to form an intermediate which may cyclize to form BM or undergo a hydrogen shift to form 3-butenal, an unstable precursor of CA. Further evidence for this mechanism was provided by the inability to detect methyl vinyl ketone, a possible product of an oxygen transfer reaction to C-2 or C-3 of 1,3-butadiene, and by the finding that CA was not simply a decomposition product of BM under assay conditions. In the second mechanism, however, chloride ion is oxidized by myeloperoxidase to HOCl which reacts with 1,3-butadiene to yield CHB. Further evidence for this mechanism was provided by the finding that CHB was readily formed when 1,3-butadiene was added to the filtrate of a myeloperoxidase/H2O2/KCl incubation and when 1,3-butadiene was allowed to react with authentic HOCl. In addition, CHB was not detected when BM or CA was incubated with myeloperoxidase, H2O2, and KCl for up to 60 min, or when 1,3-butadiene and KCl were incubated with chloroperoxidase and H2O2 or with mouse liver microsomes and NADPH, enzyme systems which catalyze 1,3-butadiene oxidation to BM and CA, but unlike myeloperoxidase, do not catalyze chloride ion oxidation to HOCl. These results provide clear evidence for novel olefinic oxidation reactions by myeloperoxidase.     

Stereospecific Biohydroxylations of Protected Carboxylic Acids with Cunninghamella blakesleeana

Cunninghamella blakesleeana DSM 1906 was found to be an efficient biocatalyst for the biotransformation of cycloalkylcarboxylic acids into hydroxy and oxo derivatives. When cultivated in submerged culture, the fungus grew in pellets. In comparison with malt extract-glucose-peptone-yeast extract medium (medium E), Czapek-Dox medium was found to reduce pellet size. Cycloalkylcarboxylic acids were protected against microbial degradation by chemical transformation into 2-cycloalkyl-1,3-benzoxazoles. The transformations of protected cyclopentyl-, cyclohexyl-, cycloheptyl-, and cyclooctylcarboxylic acids by C. blakesleeana were investigated. The biotransformations were performed in medium E by using an aerated, stirred-tank bioreactor. The transformation of 2-cyclopentyl-1,3-benzoxazole yielded (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol as the main product. The main by-product was (1R)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one, and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol was also obtained in small amounts. During the experiment, the enantiomeric excess of the main product increased up to 64%. 2-Cyclohexyl-1,3-benzoxazole was hydroxylated to 4-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol. 2-Cycloheptyl-1,3-benzoxazole and 2-cyclooctyl-1,3-benzoxazole were transformed into several alcohols and ketones, all in low yields (2 to 19%).     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.     

The biochemical toxicology of 1,3-difluoro-2-propanol,the major ingredient of the pesticide Gliftor: The potential of 4-methylpyrazole as an antidote

Administration to rats of 1,3-difluoro-2-propanol (100 mg kg⁻¹ body weight), the major ingredient of the pesticide gliftor, resulted in accumulation of citrate in the kidney after a 3 hour lag phase. 1,3-Difluro-2-propanol was found to be metabolized to 1,3-difluoroacetone and ultimately to the aconitate hydratase inhibitor (-) erythrofluorocitrate and free fluoride. The conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone was found to be catalyzed by an NAD⁺-dependent alcohol dehydrogenase, while the defluorination was attributed to microsomal monooxygenase activity induced by phenobarbitone and inhibited by piperonyl butoxide. 4-Methylpyrazole was found to inhibit both of these processes in vitro and when administered (90 mg kg⁻¹ body weight) to rats, 2 hours prior to 1,3-difluoro-2-propanol, eliminated signs of poisoning, prevented (-) erythrofluorocitrate synthesis, and markedly decreased citrate and fluoride accumulation in vivo. 4-Methylpyrazole also appeared to diminish (-) erythrofluorocitrate synthesis from fluoroacetate in vivo, and this was attributed to its capacity to inhibit malate dehydrogenase activity. The antidotal potential of 4-methylpyrazole and the potential for 1,3-difluoro-2-propanol to replace fluoroacetate (compound 1080) as a vertebrate pesticide is discussed. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 41–52, 1998     

Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.     

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.     

A light-induced β-1,3-glucan breakdown associated with the differentiation of chloroplasts in Euglena gracilis

1. Light induced a rapid breakdown of β-1,3-glucan in carbon-starved cells of Euglena gracilis, Strain Z. In contrast, β-1,3-glucan was utilized slowly in cells deprived of carbon growth substrates and maintained in the dark.

2. The breakdown of β-1,3-glucan required continuous light. At a low light intensity (7 ft candles), induction of the breakdown was delayed 12–24 h.

3. Cells showing a rapid breakdown of β-1,3-glucan contained high activities of the enzymes β-1,3-glucan phosphorylase and β-1,3-glucan hydrolase (EC 3.2.1.6) and a low activity of β-1,3-glucan synthetase (EC 2.4.1.12).

4. The light-induced breakdown of β-1,3-glucan appeared to be associated with the development of chloroplasts. A bleached mutant, ZUV-3, which cannot synthesize pigments when exposed to light, did not show a light-induced breakdown of β-1,3-glucan.

5. Chloramphenicol and 5-fluorouracil, inhibitors of the synthesis of a number of chloroplast proteins, did not inhibit the light-induced breakdown of β-1,3-glucan. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, slightly delayed the breakdown.

6. An inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), only partially inhibited chloroplast development during the period of rapid breakdown of β-1,3-glucan. After the store of β-1,3-glucan had been depleted, DCMU completely inhibited chloroplast development. The requirement for either photosynthesis or an endogenous supply of β-1,3-glucan for chloroplast development could be satisfied by supplying glucose exogenously.     

Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-d-glucan type produced by B. rhodina) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.     

Identification and characterization of three novel beta 1,3-N-acetylglucosaminyltransferases structurally related to the beta 1,3-galactosyltransferase family

We have isolated three types of cDNAs encoding novel beta1,3-N-acetylglucosaminyltransferases (designated beta3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the beta1,3-galactosyltransferase family and a beta1,3-N-acetylglucosaminyltransferase (designated beta3Gn-T1), but show no structural similarity to another type of beta1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed beta1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not beta1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with beta3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these beta3Gn-Ts were different among 35 human tissues. beta3Gn-T2 was ubiquitously expressed, whereas expression of beta3Gn-T3 and -T4 was relatively restricted. beta3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. beta3Gn-T4 was mainly expressed in brain. These results have revealed that several beta1,3-N-acetylglucosaminyltransferases form a family with structural similarity to the beta1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each beta1,3-N-acetylglucosaminyltransferase may play different roles.     

1,3-Dichloro-2-propanol induces apoptosis via both calcium and ROS in mouse melanoma cells

We demonstrated that the apoptotic cell death of mouse melanoma cells exposed to 1,3-dichloro-2-propanol (DCP) was Ca²⁺ dependent. 1,3-DCP inhibited the growth of mouse melanoma cells in a concentration-dependent manner. Furthermore, a terminal nick-end labeling of fragmented DNA (TUNEL) assay demonstrated that 1,3-DCP-induced apoptosis. Moreover, reactive oxygen species and Ca²⁺, generated by mouse melanoma cells that were exposed to 1,3-DCP, resulted in the activation of NF-κB and MAPKs. Finally, treatment with BAPTA, an intracellular Ca²⁺ chelator, completely inhibited the apoptosis that was induced by 1,3-DCP treatment of B16F10 cells.     

"Plasmalogen-type" cyclic acetals: formation and conformation of the 1,3-dioxanes and 1,3-dioxolanes from 1-0-cis-alk-1'-enyl-sn-glycerols

Acid-catalyzed cyclization of 1-O-cis-alk-1'-enyl-sn-glycerol produced four structurally and geometrically isomeric long-chain cyclic acetals of glycerol. The isomers were isolated by adsorption and gas-liquid chromatography and were identified as cis-2-alkyl-5-hydroxy-1,3-dioxane (Ia), trans-2-alkyl-5-hydroxy-1,3-dioxane (IIa), cis-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IIIa), and trans-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IVa). The structure of each isomer was established by chemical and spectroscopic methods. Cyclization with p-toluenesulfonic acid in boiling benzene led to a thermodynamically equilibrated mixture of isomers Ia-IVa in which the cis isomers predominated. Cyclization in acetic acid was found to be kinetically controlled, and formation of the trans isomers was relatively favored. Rearrangement of the cyclic acetal isomers did not occur in acetic acid; hence, optically active five-membered ring acetals were prepared.     

Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae

Production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is restrained by ethanol formation. The first step in the formation of ethanol from acetyl-CoA is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme that competes with 1,3-PD oxidoreductase for the cofactor NADH. This study aimed to improve the production of 1,3-PD by engineering the ethanol formation pathway. An inactivation mutation of the aldA gene encoding ALDH in K. pneumoniae YMU2 was generated by insertion of a tetracycline resistance marker. Inactivation of ALDH resulted in a nearly abolished ethanol formation but a significantly improved 1,3-PD production. Metabolic flux analysis revealed that a pronounced redistribution of intracellular metabolic flux occurred. The final titer, the productivity of 1,3-PD and the yield of 1,3-PD relative to glycerol of the mutant strain reached 927.6 mmol L(-1), 14.05 mmol L(-1)h(-1) and 0.699 mol mol(-1), respectively, which were much higher than those of the parent strain. In addition, the specific 1,3-PD-producing capability (1,3-PD produced per gram of cells) of the mutant strain was 2-fold that of the parent strain due to a lower growth yield of the mutant. By increasing NADH availability, this study demonstrates an important metabolic engineering approach to improve the efficiency of oxidoreduction-coupled bioprocesses.     

Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology

An operation mode with N(2) bubbling under vacuum was employed for the solvent-free synthesis of 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) from conjugated linoleic acid (CLA) catalyzed by Novozym 435. The response surface methodology (RSM) was adopted for the optimization of the reaction conditions with five major factors (incubation time, temperature, enzyme load, substrate mole ratio, and system vacuum) and three responses (CLA conversion, 1,3-dCLG yield, and acyl migration). Two sets of optimal conditions were recommended. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of 1,3-dCLG yield. Under the optimal conditions, the yield of 1,3-dCLG up to 93% was obtained. The reaction was scaled up to a production level of 100 g of 1,3-dCLG at a yield of 90.7%, indicating a promising feature of the technology in industrial applications.     

The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

Use of triphenylmethyl (trityl) amino protecting group in the synthesis of ketomethylene analogues of peptides

The Grignard reagents of 2-(2-bromoethyl)-1,3-dioxane and 2-(2-bromoethyl)-1,3-dioxolane readily reacted with the 2-thiopyridyl ester of N-triphenylmethyl-L-leucine to give the ketone adducts 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxane (8a) and 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxolane (8b) in near quantitative yield. When 1 equiv. of the Grignard reagent of 2-(2-bromoethyl)-1,3 dioxane was used, the desired ketone adduct 8a was formed slowly but quantitatively. In contrast, when 2 equiv. of the Grignard reagent were used, the formation of ketone 8a was instantaneous. The triphenylmethyl protecting group was easily removed from 8a using dilute acid to give the amino ketone 2-[3-oxo-4(S)-amino-6-methylheptyl]-1,3-dioxane oxalate salt (9). This material served as a useful intermediate in the synthesis of the ketomethylene analogues of the peptides, Z-Pro-Leu-Gly-OH and Leu-Gly-Val-Phe-OCH3.