Impact of the Cellular Zinc Status on PARP-1 Activity and Genomic Stability in HeLa S3 Cells.

Poly(ADP-ribose) polymerase 1 (PARP-1) is actively involved in several DNA repair pathways, especially in the detection of DNA lesions and DNA damage signaling. However, the mechanisms of PARP-1 activation are not fully understood. PARP-1 contains three zinc finger structures, among which the first zinc finger has a remarkably low affinity toward zinc ions. Within the present study, we investigated the impact of the cellular zinc status on PARP-1 activity and on genomic stability in HeLa S3 cells. Significant impairment of H2O2-induced poly(ADP-ribosyl)ation and an increase in DNA strand breaks were detected in the case of zinc depletion by the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) which reduced the total and labile zinc concentrations. On the contrary, preincubation of cells with ZnCl2 led to an overload of total as well as labile zinc and resulted in an increased poly(ADP-ribosyl)ation response upon H2O2 treatment. Furthermore, the impact of the cellular zinc status on gene expression profiles was investigated via high-throughput RT-qPCR, analyzing 95 genes related to metal homeostasis, DNA damage and oxidative stress response, cell cycle regulation and proliferation. Genes encoding metallothioneins responded most sensitively on conditions of mild zinc depletion or moderate zinc overload. Zinc depletion induced by higher concentrations of TPEN led to a significant induction of genes encoding DNA repair factors and cell cycle arrest, indicating the induction of DNA damage and genomic instability. Zinc overload provoked an up-regulation of the oxidative stress response. Altogether, the results highlight the potential role of zinc signaling for PARP-1 activation and the maintenance of genomic stability.

A stable isotope dilution approach to analyze ferulic acid oligomers in plant cell walls using liquid chromatography-tandem mass spectrometry.

Diferulic (DFA) and triferulic acids (TriFA) acylate and cross-link plant cell wall polysaccharides, thereby being important structural elements within the cell wall, also affecting physicochemical properties of the isolated polysaccharides. Due to the large number of potential regio- and configurational isomers and due to the fact that oligoferulic acids are not commercially available as standard compounds, analysis of oligoferulic acids after alkaline hydrolysis is challenging. Eighteen di- and triferulic acids were synthesized both non-labeled as well as 13C-labeled. By using these standard compounds, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (electrospray ionization, negative mode)-based stable isotope dilution approach was developed, fully validated and applied to plant materials. Whereas this stable isotope dilution approach is most useful to analyze plant materials with complex matrices (especially lignified tissues), less complicated matrices may not require this approach. Therefore, an alternative LC-MS/MS-based method that is based on using a single internal standard compound only was developed, too, validated, and compared to the stable isotope dilution approach. Although the stable isotope dilution approach appears to be superior, plant samples with simple matrices can also be screened by using the single internal standard method developed here.

Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways.

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.

A Multi-Step Chromatographic Approach to Purify Radically Generated Ferulate Oligomers Reveals Naturally Occurring 5-5/8-8(Cyclic)-, 8-8(Noncyclic)/8-O-4-, and 5-5/8-8(Noncyclic)-Coupled Dehydrotriferulic Acids.

Ferulate-mediated cross-linking of plant cell wall polymers has various implications on the quality of plant based food products, forage digestibility, and biomass utilization. Besides dehydrodiferulic acids (DFA), dehydrotriferulic acids (TriFA) gained increasing interest over the past two decades, because they potentially cross-link up to three polymers. Here, we describe a separation strategy to obtain several TriFA as analytical standard compounds from a reaction mixture after radical coupling of ethyl ferulate. By using silica flash chromatography, Sephadex LH-20 chromatography, and reversed phase HPLC, six known TriFA as well as three previously unidentified ferulic acid trimers were obtained, and their structures were characterized by mass spectrometry and NMR spectroscopy (1H, HSQC, COSY, HMBC, and NOESY). The novel trimers were identified as 5-5/8-8(cyclic)-, 8-8(noncyclic)/8-O-4-, and, tentatively, 5-5/8-8(noncyclic)-TriFA. Natural occurrence of these TriFA in plant cell walls was demonstrated by LC-MS/MS analyses of alkaline cell wall hydrolyzates.

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass.

Development of a QuEChERS-Based Stable-Isotope Dilution LC-MS/MS Method To Quantitate Ferulic Acid and Its Main Microbial and Hepatic Metabolites in Milk.

Forage plants of the Poaceae family are grown as pasturage or used for the production of hay, straw, corn stover, etc. Although ferulic acid contents of grasses are generally high, the amount of ingested ferulic acid differs depending on the type of forage, resulting in varying contents of ferulic acid and its microbial and hepatic metabolites in milk. Concentrations and patterns of these metabolites may be used as markers to track different forages in livestock feeding. Therefore, we developed a stable isotope dilution assay to quantitate ferulic acid, 12 ferulic acid-based metabolites, p-coumaric acid, and cinnamic acid in milk. Because most analytes were not commercially available as stable isotope labeled standard compounds, they were synthesized as 13C- or deuterium-labeled standard compounds. A modification of the QuEChERS method, a Quick, Easy, Cheap, Effective, Rugged, and Safe approach usually applied to analyze pesticides in plant-based products, was used to extract the phenolic acids from milk. Determination was carried out by LC-ESI-MS/MS in scheduled multiple reaction monitoring modus. By using three different milk samples, the applicability of the validated approach was demonstrated.

Identification of 8-O-4/8-5(Cyclic)- and 8-8(Cyclic)/5-5-Coupled Dehydrotriferulic Acids, Naturally Occurring in Cell Walls of Mono- and Dicotyledonous Plants.

Besides ferulate dimers, higher oligomers of ferulic acid such as trimers and tetramers were previously demonstrated to occur in plant cell walls. This paper reports the identification of two new triferulic acids. 8-O-4/8-5(cyclic)-triferulic acid was synthesized from ethyl ferulate under oxidative conditions using copper(II)-tetramethylethylenediamine [CuCl(OH)-TMEDA] as a catalyst, whereas 8-8(cyclic)/5-5-triferulic acid was isolated (preparative size exclusion chromatography, reversed-phase HPLC) from saponified insoluble maize fiber. Structures of both trimers were unambiguously elucidated by high-resolution LC-ToF-MS/MS and one- ((1)H) and two-dimensional (HSQC, HMBC, COSY, NOESY) NMR spectroscopy. The newly described trimers were identified by LC-MS/MS in alkaline hydrolysates of insoluble fibers from maize, wheat, and sugar beet, indicating that ferulic acid cross-links between cell wall polymers are more diverse than previously recognized. Saponification experiments also suggest that the newly identified 8-O-4/8-5(cyclic)-triferulic acid is the naturally occurring precursor of the previously identified 8-O-4/8-5(noncyclic)-triferulic acid in plant cell walls.