Polycytidylate and poly(7-deazaguanylate): A pair of complementary templates

We have studied the template-directed oligomerization on polycytidylic acid of the 5-phosphoro(2-methyl)imidazolides of a number of analogues of guanosine. None of the analogues reacted as efficiently as the original guanosine compound, and only the 7-deazaguanosine analogue gives a detectable yield of oligomers. Similar results are described for a reaction involving the intramolecular template-directed elongation of a short oligocytidylate primer.Oligocytidylates containing five or more cytidylate residues are extended on the single-stranded regions of poly(G). In the present study we show that these oligocytidylates are extended efficiently by reaction with cytidine-5-phosphoro(2-methyl) imidazolide on a poly(7-deazaguanylic acid) template. The products are considerably longer than those obtained using a polyguanylic acid template. We believe that the formation of a tetrahelix inhibits the latter reaction, while poly(7-deazaguanylate) does not aggregate and, therefore, acts as a more efficient template. This work identifies for the first time a pair of homopolymers each of which facilitates the template-directed elongation of the other.Deceased Correspondence to: L.E. Orgel     

10-Hydroxy-8(Z)-octadecenoic acid,an intermediate in the bioconversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid

Summary Previously, we reported the discovery of a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) which was produced from oleic acid by a new bacterial isolate PR3 [6,7]. The reaction is unique in that it involves a hydroxylation at two positions and a rearrangement of the double bond of the substrate molecule. Now, we have isolated another compound from the reaction mixture determined by GC/MS to be 10-hydroxy-8-octadecenoic acid (HOD). NMR and IR data indicate that the unsaturation is probablycis. The optimum pH and temperature for the production of HOD by strain PR3 were 6.5 and 30°C, about the same as those for DOD. However, the amount of HOD detected remained small throughout an 48-h reaction period during which the amount of DOD increased sharply. At 48 h of reaction, the ratio between HODDOD was 110. HOD may be an intermediate in the biosynthesis of DOD from oleic acid.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Raman spectroscopic measurements in self‐pressurized aqueous solutions above 100°C: The melting of poly(G) and poly(G) · poly(C)

We have studied by Raman spectroscopy the thermal behavior of associated polyguanylic acid [poly(G)] and polyguanylic–polycytidylic acid [poly(G) · poly(C)] in self‐pressurized aqueous solutions contained in sealed capillary tubes. The associated polynucleotides were found to be very resistant to heat, but evidence of thermal degradation was observed after melting of the helical structures. The cooperative melting transition of the four‐stranded complex of poly(G) was located at 141°C in 0.5M KCl, 135°C in 0.5M NaCl, 129°C in 0.5M LiCl, 123°C in 0.1M tetramethylammonium perchlorate, and 105°C in 0.1M tetraethylammonium bromide solutions. The transition was observed at 130°C in poly(G) · poly(C) (in 0.5M NaCl). The results in this case show that a four‐stranded poly(G) complex is formed following the melting of the double helix. © 1999 John Wiley & Sons, Inc. Biopoly 49: 21–28, 1999     

Enzymatic Production of Pyrimidine Nucleotides Using Corynebacterium ammoniagenes Cells and Recombinant Escherichia coli Cells: Enzymatic Production of CDP-Choline from Orotic Acid and Choline Chloride (Part I)

Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mm) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mm) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mm) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.     

Facile synthesis of (R)N-2-hydroxyacyl-L-cysteine derivatives: (R)N-2-hydroxyacyl transfer from enzymatically-synthesized (R)S-2-hydroxyacylglutathione derivatives to L-cysteine

Summary N-(R)-2-Hydroxyacyl-L-cysteine derivatives were conveniently synthesized by the reaction of the corresponding S-(R)-2-hydroxyacyl-glutathione with cysteine. The (R)2-hydroxyacyl group was transferred from the S-glutathionyl moiety to S-cysteinyl, forming the corresponding (R)S-2-hydroxyacylcysteine; this rearranged to the (R)N-hydroxyacylcysteine. These compounds have anti-proliferative activity associated with the inhibition ofde novo pyrimidine synthesis.Abbreviations TRIS tris(hydroxymethyl) aminomethane - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Which 3-ribofuranosyl-substituted purine 5′-phosphates undergo template-directed oligomerization?

Summary We have studied the oligomerization reactions of the 2-methylimidazolide derivatives of 3-isoisoguanosine 5-phosphate (2) and 3-isoxanthosine 5-phosphate (5) in the presence of a variety of homopolynucleotide templates. In no case did we observe a substantial template-facilitated production of long oligomers. Polyuridylic acid directed the synthesis of low molecular-weight products from both monomers. Polycytidylic acid, polyadenylic acid, polyinosinic acid, and polyguanylic acid were ineffective as templates in the systems that we investigated.     

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Nitric oxide (NO) is thought to react with fatty acid alkoxyl radical, which is generated from fatty acid hydroperoxide via one-electron reduction. However, detail in the reaction remains obscure. In the present study, we examined the reaction of nitric oxide with fatty acid alkoxyl radical generated in the lipoxygenase/linoleate/13-hydroperoxyoctadecadienoate (13-HpODE) system under anaerobic conditions via HPLC equipped with mass spectrometry and photodiode array detections. In this reaction system, nitric oxide can scavenge linoleate alkoxyl radical, producing 13-ONO-9Z,11E-ODE. However, instead of 13-ONO-9Z,11E-ODE, 13-NO-9E,11E-ODE and 9-NO-10E,12E-ODE were alternatively detected in the reaction solution. To explain this contradiction, we proposed a mechanism as follows: (1) 13-ONO-9E/11Z-ODE undergoes homolytic cleavage at >CHONO bond into the linoleate allyl radical and nitrogen dioxide, (2) the allyl radical undergoes resonance stabilization into the E/E-form, and (3) nitric oxide scavenges the E/E-pentadiene radical at C9 or C13 position. Consequently, we concluded that nitric oxide immediately converts fatty acid alkoxyl radical into allyl radical.     

Formation of (n-9) and (n-7) cis-monounsaturated fatty acids in seeds of higher plants

The relative abundance of (n-9) and (n-7) isomers in the monounsaturated fatty acids of seed lipids has been determined for selected plants in order to assess the biosynthetic reactions involved in their formation. 9 Desaturation of stearic acid to (n-9) octadecenoic acid is almost exclusively operative in the formation of monounsaturated fatty acids in the seeds of Helianthus annuus, Glycine max and Brassica napus, cv. Quinta and Erglu, in which chain elongation of monounsaturated fatty acids terminates at the level of an 18 carbon chain. 9 Desaturation of palmitic acid is a minor yet significant pathway in the seeds of Sinapis alba and Brassica napus, cv. Rapol and Tira, in which chain elongation of monounsaturated fatty acids occurs extensively beyond the 18 carbon chain. In each of these seeds, both (n-9) and (n-7) octadecenoic acids formed are subsequently elongated to icosenoic acids. However, elongation of the (n-7) isomer is terminated at the level of a 20 carbon chain, whereas the (n-9) icosenoic acid is selectively elongated to docosenoic acid and even up to tetracosenoic acid in Sinapis alba. 9 Desaturation of palmitic acid followed by elongation to (n-7) octadecenoic acid occurs to a minor extent in the seeds of Tropaeolum majus. Only the (n-9) octadecenoic acid, and not its (n-7) isomer, is elongated to icosenoic and docosenoic acids.     

Fatty Acid Composition of Mitochondrial Membrane Lipids in Cultivated (Zea mays) and Wild (Elymus sibiricus) Grasses

The fatty acid composition of mitochondrial membrane lipids from seedlings of two grass species differing in their chilling tolerance, elymus (Elymus sibiricus) and maize (Zea mays), was studied by gas-liquid chromatography. An increased chilling tolerance of elymus, a perennial wild species, seems to be caused by a high content of unsaturated fatty acid residues in the total membrane lipids; linoleic (41.9%) and -linolenic (23.4%) acids predominated among these fatty acids. The contents of linoleic and -linolenic acids in the total lipids of maize membranes were 57.5% and 5.8%, respectively. Polar lipids of elymus mitochondrial membranes were characterized by about similar contents of linoleic (33.6%) and -linolenic (31.3%) acids. Linoleic acid (51.7%) predominated in maize mitochondrial polar lipids, whereas the -linolenic acid content was 9.0%. A high level of 3-desaturase activity in the mitochondrial membranes seems to be an important factor of elymus chilling tolerance. This high activity seems to be developed as an adaptation in the course of evolution of this species.     

Construction of a non-support bioreactor: optical resolution of 2-(4-chlorophenoxy)propanoic acid in an organic solvent system

Summary A non-support bioreactor, a novel column reactor packed with a free non-supported enzyme was constructed by applying the insolubility of the enzyme in organic solvents. Stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid by lipase OF 360 from Candida cylindracea with n-tetradecanol was selected as a model reaction. Non-supported lipase revealed threefold higher activity than Celite-adsorbed lipase by maintaining high stereoselectivity in a batch reaction. In continuous operation, a non-support bioreactor produced the ester with fourfold higher productivity to that of a column reactor packed with Celite-adsorbed lipase (an adsorbed bioreactor). However, the optical purity of the remaining (S)-acid was low even when the conversion ration was kept at approximately 50%. Lipase recovered from the non-support bioreactor after continuous operation retained the original stereoselectivity in a batch reaction. Therefore, semi-continuous operation was conducted by recycling the substrate solution at a high flow rate. The non-support reactor showed high stereoselectivity and ten times the productivity compared with the adsorbed bioreactor. The reason for this high performance is discussed. Offprint requests to: A. Tanaka     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and beta-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via -oxidation. However, -oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain -oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as -oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain -oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous -oxidation by glyoxysomes.     

Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3") (9)

Summary The nucleotide sequence of the spc determinant of the Staphylococcus aureus transposon Tn554 has been determined. This gene encodes a spectinomycin adenyltransferase, AAD(9), that mediates resistance to spectinomycin but not to streptomycin. The sequence predicts a 260 amino acid protein of molecular weight 28,943. A spectinomycin-sensitive mutant (spc-1) contains a GA transition resulting in substitution of threonine (ACA) for alanine (GCA) at residue 165. The predicted amino acid sequence is 36% homologous to that of a widely distributed, gramnegative streptomycin/spectinomycin adenyltransferase, AAD(3) (9), specified by the aadA determinant (Holingshead and Vapnek 1985).     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

Enzymatic synthesis and characterization of N5-(carboxymethyl)-L-ornithine and N6-(carboxymethyl)-L-lysine

Summary This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N⁶-(carboxymethyl)-L-lysine and N⁵-(carboxymethyl)-L-ornithine. The two N -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and the- or-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N⁵-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.     

Auxin,biosynthesis of ethylene and sex expression in cucumber (Cucumis sativus)

Ethylene production, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and activity of the ethylene forming enzyme (EFE) were higher in apices of gynoecious cucumber (Cucumis sativus cv. Alma) as compared to monoecious cucumber (C. sativus cv. Elem). Application of indole-3-acetic acid (IAA) enhanced ethylene and ACC production in both cultivars. The stimulatory effect of IAA was more pronounced in gynoecious apices. Induction of ethylene production and accumulation of ACC resulting from treatment with IAA were effectively blocked by aminoethoxyvinylglycine (AVG). Content of endogenous IAA, measured by an enzyme immunoassay, was lower in gynoecious cucumber as compared to monoecious one. Treatment of gynoecious plants with the antiauxins -(p-chlorophenoxy)isobutyric acid (PCIB) and -naphthaleneacetic acid (-NAA) did not inhibit female sex expression.It appears that although exogenous IAA enhances ACC and ethylene production, endogenous IAA might not have a major role in the control of sex expression in cucumber of the Beit-Alfa type.Prof. Rudich passed away in May 1986.     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by sodium (2,2'-bipyridine) oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5 regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.Abbreviations 2,4-D 2,4 Dichlorophenoxyacetic acid - GUS Glucuronidase - Hm Hygromycin - HPH Hygromycin phosphotransferase - MS medium Murashige and Skoog medium - PCR Polymerase chain reaction - X-Gluc 5-Bromo-4-chloro--indolyl--D-glucuronic acid