Degradation of cytokinins by maize cytokinin dehydrogenase is mediated by free radicals generated by enzymatic oxidation of natural benzoxazinones

Hydroxamic acid 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐one (DIMBOA) was isolated from maize phloem sap as a compound enhancing the degradation of isopentenyl adenine by maize cytokinin dehydrogenase (CKX), after oxidative conversion by either laccase or peroxidase. Laccase and peroxidase catalyze oxidative cleavage of DIMBOA to 4‐nitrosoresorcinol‐1‐monomethyl ether (coniferron), which serves as a weak electron acceptor of CKX. The oxidation of DIMBOA and coniferron generates transitional free radicals that are used by CKX as effective electron acceptors. The function of free radicals in the CKX‐catalyzed reaction was also verified with a stable free radical of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid. Application of exogenous cytokinin to maize seedlings resulted in an enhanced benzoxazinoid content in maize phloem sap. The results indicate a new function for DIMBOA in the metabolism of the cytokinin group of plant hormones.     

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4‐benzoxazin‐3‐ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4‐benzoxazin‐3‐ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4‐benzoxazin‐3‐ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4‐benzoxazin‐3‐ones and active extracts from mutant plants lacking 1,4‐benzoxazin‐3‐ones. Our data show that maize plants possess a two‐step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2‐β‐d ‐glucopyranosyloxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA‐Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA) is released by plant‐derived β‐glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4‐benzoxazin‐3‐ones such as 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.     

Chiral separation of pheniramine-like 3-phenyl-3-heteroarylpropylamines by CE and HPLC methods

Analytical CE and HPLC methods were developed for the chiral separation of halogen-substituted 3-phenyl-3-(2-pyridyl)propylamines 1-4 (1: 3-(4-fluorophenyl) approximately, 2: 3-(3,4-difluorophenyl) approximately, 3: 3-(4-chlorophenyl) approximately, 4: 3-(3,4-dichlorophenyl) approximately ), 3-(4-fluorophenyl)-3-(2-thiazolyl)propylamine (5), and 3-(4-fluorophenyl)-3-(1-benzylimidazol-2-yl)propylamine (6), which are building blocks for the preparation of very potent arpromidine-type histamine H(2) receptor agonists. All amines were enantioseparated by CE with resolutions of at least 1.8 using alpha-, beta-, or gamma-cyclodextrin (CD) as chiral selectors. With heparin as buffer additive for CE the optical antipodes of 1-4 and 6 were separated with resolutions > or = 1.8. On RP-18 columns the separation of the (+)-(S)-acetylmandelic acid amides of racemic 2 (R = 0.9, alpha = 1.07) and the thioureas prepared by addition of 6 to 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (R = 2.0, alpha = 1.20) was successful, whereas the diastereomeric ureas prepared from 3 and (+)-(S)-1-(1-naphthyl)ethyl isocyanate could not be resolved. Separation of the diastereomeric isoindoles prepared from 1-5, o-phthaldialdehyde and 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside was achieved on a RP-18 phase (R > or = 0.4, a > or = 1.02). Direct separation of the enantiomers of 3 and 4 was achieved on a Cyclobond I column (R > or = 0.9, alpha > or = 1.07). alpha- and beta-CD were also useful as mobile phase additives for HPLC (3 and 4: RP-18 column, beta-CD, R > or = 0.4, alpha > or = 1.03; 3: RP-18 column, alpha-CD: R = 0.5, alpha = 1.04).     

Enantioselective synthesis of (S)-2-amino-4-phenylbutanoic acid by the hydantoinase method

Biosynthesis of (S)-(+)-2-amino-4-phenylbutanoic acid (1) was performed by nonenantioselective hydantoinase and L-N-carbamoylase using racemic 5-[2-phenylethyl]-imidazolidine-2,4-dione (rac-2) as a substrate. The compounds involved in this biocatalysis process could be simultaneously resolved by high-performance liquid chromatography using Chirobiotic T column with a mobile phase of EtOH/H(2)O = 10/90 at pH 4.2-4.5. To our knowledge, this is the first report of the successful production of 1 by the combination of recombinant hydantoinase and L-N-carbamoylase.     

Enantioseparation of chiral alcohols by complex formation and subsequent supercritical fluid extraction

The very first application of supercritical fluid extraction (SFE) on enantioseparation of alcohols is discussed. Resolution of three chiral alcohols (trans-2-chloro-cyclohexanol, trans-2-bromo-cyclohexanol, and trans-2-iodo-cyclohexanol) were performed by partial complexation with (-)-O,O'-dibenzoyl-(2R,3R)-tartaric acid monohydrate (DBTA). DBTA formed diastereomeric complexes with all S,S-enantiomers stable enough to extract the unreacted alcohols with supercritical carbon dioxide. Resolution efficiency increased with the size of halogen substituents, and by the proper selection of molar ratio, pure (-)-R,R-trans-2-iodo-cyclohexanol (ee > 99%, yield: 39%) or (+)-S,S-trans-2-iodo-cyclohexanol (ee = 98%, yield: 8%) were prepared in one process step. Achieved resolution efficiency values were much higher in all resolution procedures than in any other known enantioseparation of these racemic compounds. The developed method offers an environmentally friendly, efficient alternative of currently applied resolution processes, also on a preparative scale.     

Gene stacking by recombinases

Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site‐specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high‐efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.     

Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for the isolation and purification of 1'-O-glucosylcimifugin (1), 4'-O-beta-d-glucosyl-5-O-methylvisamminol (2), cimifugin (3) and 3'-O-glucosylhamaudol (4) from the Chinese medicinal herb radix saposhnikoviae has been successfully developed. A sample of 300 mg of crude extract was separated using ethyl acetate:n-butanol:1% aqueous acetic acid (1:4:5, v/v) as the two-phase solvent system and yielded 102.4 mg of 1 and 81.6 mg of 2. During this separation 3 and 4 remained in the stationary phase, which was collected, evaporated to dryness and separated with another two-phase solvent system involving ethyl acetate:n-butanol:1% aqueous acetic acid (5:0.5:5, v/v) to yield 31.4 mg of 3 and 12.7 mg of 4. The purities of compounds 1-4 were 98.4, 98.7, 99.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were established by (1)H-NMR and (13)C-NMR.     

Enantioselective Aldol Reaction Between Isatins and Cyclohexanone Catalyzed by Amino Acid Sulphonamides

Sulphonamides derived from primary α‐amino acid were successfully applied to catalyze the aldol reaction between isatin and cyclohexanone under neat conditions. More interestingly, molecular sieves, as privileged additives, were found to play a vital role in achieving high enantioselectivity. Consequently, high yields (up to 99%) along with good enantioselectivities (up to 92% ee) and diastereoselectivities (up to 95:5 dr) were obtained. In addition, this reaction was also conveniently scaled up, demonstrating the applicability of this protocol. Chirality 27:314‐319, 2015. © 2015 Wiley Periodicals, Inc.     

One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis

A novel one-pot procedure for the nonreductive release of O-linked glycans from glycoproteins and the simultaneous derivatization of released glycans with 1-phenyl-3-methyl-5-pyrazolone (PMP) is described. Unlike the traditional reductive β-elimination, which produces alditols, this new method employs PMP/ammonia aqueous solution as the reaction medium. The O-glycans are released from glycoproteins and derivatized with PMP nonreductively, specifically, and quantitatively. Samples can be easily purified from ammonia, excess PMP, and peptide residues by evaporation, chloroform extraction, and solid-phase extraction (SPE) column fractionation for HPLC, CE, or MS analysis. The procedure has been elaborated with two purified glycoproteins, porcine stomach mucin and bovine fetuin, and successfully applied to O-glycan profiling of a challenging biological specimen, healthy human plasma. This new procedure has shown methodological significance in O-glycan analysis.     

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.     

Predicting protein quaternary structure by pseudo amino acid composition

In the protein universe, many proteins are composed of two or more polypeptide chains, generally referred to as subunits, that associate through noncovalent interactions and, occasionally, disulfide bonds. With the number of protein sequences entering into data banks rapidly increasing, we are confronted with a challenge: how to develop an automated method to identify the quaternary attribute for a new polypeptide chain (i.e., whether it is formed just as a monomer, or as a dimer, trimer, or any other oligomer). This is important, because the functions of proteins are closely related to their quaternary attribute. For example, some critical ligands only bind to dimers but not to monomers; some marvelous allosteric transitions only occur in tetramers but not other oligomers; and some ion channels are formed by tetramers, whereas others are formed by pentamers. To explore this problem, we adopted the pseudo amino acid composition originally proposed for improving the prediction of protein subcellular location (Chou, Proteins, 2001; 43:246-255). The advantage of using the pseudo amino acid composition to represent a protein is that it has paved a way that can take into account a considerable amount of sequence-order effects to significantly improve prediction quality. Results obtained by resubstitution, jack-knife, and independent data set tests, have indicated that the current approach might be quite promising in dealing with such an extremely complicated and difficult problem.     

Antifungal metabolites from the roots of Diospyros virginiana by overpressure layer chromatography

A preparative overpressure layer chromatography (OPLC) method was successfully used for the separation of two new natural compounds, 4‐hydroxy‐5,6‐dimethoxynaphthalene‐2‐carbaldehyde ( 1 ) and 12,13‐didehydro‐20,29‐dihydrobetulin ( 2 ) together with nine known compounds, including 7‐methyljuglone ( 3 ), diospyrin ( 4 ), isodiospyrin ( 5 ), shinanolone ( 6 ), lupeol ( 7 ), betulin ( 8 ), betulinic acid ( 9 ), betulinaldehyde ( 10 ), and ursolic acid ( 11 ) from the acetone extract of the roots of Diospyros virginiana. Their identification was accomplished by 1D‐ and 2D‐NMR spectroscopy and HR‐ESI‐MS methods. All the isolated compounds were evaluated for their antifungal activities against Colletotrichum fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and P. viticola using in vitro micro‐dilution broth assay. The results indicated that compounds 3 and 5 showed high antifungal activity against P. obscurans at 30 μM with 97.0 and 81.4% growth inhibition, and moderate activity against P. viticola (54.3 and 36.6%). It appears that an optimized OPLC system offers a rapid and efficient method of exploiting bioactive natural products.     

Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro-organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG-treated seedlings. 2-D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2-D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.     

Synthesis of 3-tert-butylcatechol by an engineered monooxygenase

Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.     

Scavenging of hydroxyl radical by catecholamines

The direct effects of the four catecholamines (CATs), adrenaline (A), noradrenaline (NA), dopamine (D) and isoproterenol (I), on free radicals were investigated using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) and hydroxyl radial (HO^?). The CATs examined were found to inhibit the ESR signal intensity of DPPH^? in a dose‐dependent manner over the range 0.1–2.5 mmol/L in the following order: NA > A > I > D, with IC₅₀ = 0.30 ± 0.03 for noradrenaline and IC₅₀ = 0.86 ± 0.02 for dopamine. Hydroxyl radicals were produced using a Fenton reaction in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), and ESR technique was applied to detect the CATs reactivity toward the radicals. The reaction rates constant (k_r) of CATs with HO^? were found to be in the order of 10⁹ L/mol/s, and the k_r value for noradrenaline was the highest (k_r = 8.4 × 10⁹ L/mol/s). The CATs examined exhibited also a strong decrease in the light emission (62–73% at 1 mmol/L concentration and 79–89% at 2 mmol/L concentration) from a Fenton‐like reaction. These reactions may be relevant to the biological action of these important polyphenolic compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.