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.     

Practical asymmetric synthetic route to 4,4,4-trifluoro-3-hydroxybutyrate: head-to-tail and head-to-head crystallizations through double and single hydrogen bonds of hetero- and homochiral 4,4,4-trifluoro-3-hydroxybutyrophenones

A practical asymmetric synthetic route to 4,4,4-trifluoro-3-hydroxybutyrophenone and the butyric acid phenyl ester is described using heterochiral crystallization through double hydrogen bonding assembly in head-to-tail fashion and sequential Baeyer-Villiger oxidation reaction by trifluoroperacetic acid.     

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).     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Structural and spectroscopic analysis of hydrogensquarates of glycine-containing tripeptides

The hydrogensquarates of glycine-containing tripeptides glycylglycylglycine (H-Gly-Gly-Gly-OH), glycylglycylmethionine (H-Gly-Gly-Met-OH), and methionylglycylglycine (H-Met-Gly-Gly-OH) have been characterized structurally. Quantum chemical ab initio calculations, solid-state linear-dichroic infrared (IR-LD) spectroscopy, 1H and 13C NMR data, ESI-MS, HPLC-MS/MS, TGV, and DSC methods were employed. The structures consist in a positively charged peptide moiety and a negative hydrogensquarate anion (HSq), stabilized by strong intermolecular hydrogen bonds.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

Chemical constituents of Papulaspora immersa, an endophyte from Smallanthus sonchifolius (Asteraceae), and their cytotoxic activity

Papulaspora immersa H. H. Hotson was isolated from roots and leaves of Smallanthus sonchifolius (Poepp. and Endl. ) H. Rob. (Asteraceae), traditionally known as Yacon. The fungus was cultured in rice, and, from the AcOEt fraction, 14 compounds were isolated. Among them, (22E,24R)‐8,14‐epoxyergosta‐4,22‐diene‐3,6‐dione ( 4 ), 2,3‐epoxy‐1,2,3,4‐tetrahydronaphthalene‐c‐1,c‐4,8‐triol ( 10 ), and the chromone papulasporin ( 13 ) were new secondary metabolites. The spectral data of the known natural products were compared with the literature data, and their structures were established as the (24R)‐stigmast‐4‐en‐3‐one ( 1 ), 24‐methylenecycloartan‐3β‐ol ( 2 ), (22E,24R)‐ergosta‐4,6,8(14),22‐tetraen‐3‐one ( 3 ), (?)‐(3R,4R)‐4‐hydroxymellein ( 5 ), (?)‐(3R)‐5‐hydroxymellein ( 6 ), 6,8‐dihydroxy‐3‐methylisocoumarin ( 7 ), (?)‐(4S)‐4,8‐dihydroxy‐α‐tetralone ( 8 ), naphthalene‐1,8‐diol ( 9 ), 6,7,8‐trihydroxy‐3‐methylisocoumarin ( 11 ), 7‐hydroxy‐2,5‐dimethylchromone ( 12 ), and tyrosol ( 14 ). Compound 4 showed the highest cytotoxic activity against the human tumor cell lines MDA‐MB435 (melanoma), HCT‐8 (colon), SF295 (glioblastoma), and HL‐60 (promyelocytic leukemia), with IC₅₀ values of 3.3, 14.7, 5.0 and 1.6 μM , respectively. Strong synergistic effects were also observed with compound 5 and some of the isolated steroidal compounds.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

Microwave-assisted synthesis of 4'-azaflavones and their N-alkyl derivatives with biological activities

4'-Azaflavone (=2-(pyridin-4-yl)-4H-1-benzopyran-4-one; 4) and 3-[(pyridin-4-yl)methyl]-4'-azaflavone (5) were synthesized by a simple environmentally friendly microwave-assisted one-pot method through the cyclization of 3-hydroxy-1-(2-hydroxyphenyl)-3-(pyridin-4-yl)propan-1-one (1), (E)-2'-hydroxy-4-azachalcone (2; chalcone=1,3-diphenylprop-2-en-1-one), and 2'-hydroxy-2-[(hydroxy)(pyridin-4-yl)methyl]-4'-azachalcone (3) under solventless conditions using silica-supported NaHSO(4), followed by treatment with base. In addition, N-alkyl-substituted 4'-azaflavonium bromides 6 and 7 were prepared from compounds 4 and 5, respectively. The antimicrobial and antioxidant activities of compounds 1-7 were tested. The N-alkyl-substituted 4'-azaflavonium bromides 6 and 7 showed high antimicrobial activity against the Gram-positive bacteria and the fungus tested, with MIC values close to those of reference antimicrobials ampicilline and fluconazole. The alkylated compounds 6 and 7 also showed a good antioxidant character in the two antioxidant methods, DPPH (=1,1-diphenyl-2-picrylhydrazyl) radical-scavenging and ferric reducing/antioxidant power (FRAP) tests.     

Novel triterpenoid acyl esters and alkaloids from Anoectochilus roxburghii

Two novel sorghumol acyl esters, sorghumol 3-O-Z-p-coumarate and sorghumol 3-O-E-p-coumarate, and a novel alkaloid, anoectochine, were isolated from the whole plants of Anoectochilus roxburghii along with one known triterpenoid, sorghumol. Their structures were established by their detailed spectral studies, including two-dimensional NMR ((1)H-(1)H COSY, HSQC and HMBC).     

Dynamic change and derivation process of FC and DFC through G1, S and G2 phases in HeLa cells

In order to get a deeper understanding of the relationship between nucleolus structure and its function, the dynamic change and derivation of FC (fibrillar center) and DFC (dense fibrillar component) through interphase were investigated in HeLa cells synchronized at the ultrastructural level. The results showed that there was a process of FC and DFC derivation in the nucleolus of HeLa cells during interphase. In G1 phase there were a few big FCs in the nucleolus of the HeLa cell. In S phase DFC around the FC got thickened and the configuration of the DFC changed. A lot of tiny FCs were derived from parts of the thickened DFC. We called the FC and DFC formed in G1 phase as primary FC (pri-FC) and primary DFC (pri-DFC) and the FC and DFC derived from the thickened pri-DFC as secondary FC (sec-FC) and secondary DFC (sec-DFC). In G2 phase sec-FC and sec-DFC were gradually separated from pri-DFC and scattered evenly in the nucleolus. Few large pri-FCs coexisted with numerous tiny sec-FCs in the nucleolus of HeLa cells in G2 phase. Based on the results of our observation, we suggest here a model of the dynamic change and the process of derivation of FC and DFC through interphase.     

Anthropometric Indices and the Incidence of Hypertension: A Comparative Analysis

Objective: To investigate the association between several anthropometric measurements of obesity with the incidence of hypertension. Research Methods and Procedures: Participants were 592 individuals free of hypertension, selected at random from the community. In the baseline evaluation, they were submitted to completed measures of demographics, anthropometrics, blood pressure, and other risk factors for hypertension. Incident hypertension was defined by blood pressure equal or higher than 140/90 mm Hg or use of blood pressure‐lowering drugs. Results: During a mean follow‐up time of 5.6 ± 1.1 years, 127 developed hypertension. The hazard ratios for the development of hypertension, adjusted for age, baseline blood pressure, gender, and alcohol consumption, were 1.042 (p = 0.091) for BMI, 1.023 (p = 0.028) for waist circumference, 1.042 (p = 0.013) for waist‐to‐height ratio, 1.061 (p = 0.014) for waist‐to‐height² index, 1.079 (p = 0.022) for waist‐to‐height³ index, and 1.033 (p = 0.006) for the waist‐to‐hip ratio. Discussion: The correction of the circumference of waist for stature or hip circumference improves its performance in the prediction of the incidence of hypertension.     

Biosynthetic pathway to neuromelanin and its aging process

Using model compounds of the melanic component of neuromelanin (NM) prepared by tyrosinase oxidation at various ratios of dopamine (DA) and cysteine (Cys) under physiological conditions, we examined a biosynthetic pathway to NM and its aging process by following the time course of oxidation to NM and the subsequent structural modification of NM under various heating conditions. Chemical degradation methods were applied to the synthetic NM. 4‐Amino‐3‐hydroxyphenylethylamine (4‐AHPEA) and thiazole‐2,4,5‐tricarboxylic acid (TTCA) were used as markers of benzothiazine and benzothiazole units, respectively. By following the time course of the biosynthetic pathway of synthetic NM, we found that neurotoxic molecules are trapped in NM. An aging simulation of synthetic NM showed that benzothiazine units in NM are gradually converted to benzothiazole during the aging process. Thus, natural NM was found to be similar to aged (heated) NM prepared from a 2:1 molar ratio of DA and Cys.     

Uptake and metabolism of d‐glucose in isolated acinar and ductal cells from rat submandibular glands

The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d ‐glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d ‐[U‐¹⁴C]glucose and its non‐metabolised analogue 3‐O‐[¹⁴C‐methyl]‐d ‐glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d ‐glucose and 3‐O‐methyl‐d ‐glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d ‐glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d ‐glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca²⁺ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin‐sensitive, cytochalasin B‐sensitive and phloridzin‐sensitive transport of d ‐glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd.     

Experimental conformational energy maps of proteins and peptides

We have presented an extensive analysis of the peptide backbone dihedral angles in the PDB structures and computed experimental Ramachandran plots for their distributions seen under a various constraints on X‐ray resolution, representativeness at different sequence identity percentages, and hydrogen bonding distances. These experimental distributions have been converted into isoenergy contour plots using the approach employed previously by F. M. Pohl. This has led to the identification of energetically favored minima in the Ramachandran (? , ψ ) plots in which global minima are predominantly observed either in the right‐handed α‐helical or the polyproline II regions. Further, we have identified low energy pathways for transitions between various minima in the (? ,ψ ) plots. We have compared and presented the experimental plots with published theoretical plots obtained from both molecular mechanics and quantum mechanical approaches. In addition, we have developed and employed a root mean square deviation (RMSD) metric for isoenergy contours in various ranges, as a measure (in kcal.mol^?1) to compare any two plots and determine the extent of correlation and similarity between their isoenergy contours. In general, we observe a greater degree of compatibility with experimental plots for energy maps obtained from molecular mechanics methods compared to most quantum mechanical methods. The experimental energy plots we have investigated could be helpful in refining protein structures obtained from X‐ray, NMR, and electron microscopy and in refining force field parameters to enable simulations of peptide and protein structures that have higher degree of consistency with experiments. Proteins 2017; 85:979–1001. © 2017 Wiley Periodicals, Inc.     

G-protein-coupled receptors and melanoma

G-protein-coupled receptors (GPCR) are the largest family of receptors with over 500 members. Evaluation of GPCR gene expression in primary human tumors identified over-expression of GPCR in several tumor types. Analysis of cancer samples in different disease stages also suggests that some GPCR may be involved in early tumor progression and others may play a critical role in tumor invasion and metastasis. Currently, >50% of drug targets to various human diseases are based on GPCR. In this review, the relationships between several GPCR and melanoma development and/or progression will be discussed. Finally, the possibility of using one or more of these GPCR as therapeutic targets in melanoma will be summarized.     

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.     

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.     

Caspase inhibition and N6-benzyladenosine-induced apoptosis in HL-60 cells.

As an extension of our recently published work (Mlejnek and Kuglík [2000] J. Cell. Biochem. 77:6-17), the role of caspases in N(6)-benzylaminopurine riboside (BAPR)-induced apotosis in HL-60 cells was evaluated in this study. Here, BAPR-induced apoptosis was accompanied by activation of caspase-3 and caspase-9. However, when these caspases were selectively inhibited, the progression of BAPR-induced apoptosis was not markedly affected. Besides that, activation of caspase-3 and caspase-9 was found to be rather late event in apoptotic process. These results suggested that other caspases might be critically implicated. Indeed, pan-specific caspase inhibitor, Z-VAD-FMK, completely prevented DNA cleavage and apoptotic bodies formation. However, Z-VAD-FMK failed to prevent cell death and it was incapable to fully counteract the main apoptotic hallmark-chromatin condensation. Finally, our data indicate that cellular decision between apoptosis and necrosis is made upon the availability of both caspase proteases and intracellular ATP.