Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.     

Multiobjective evaluation of advanced manufacturing system technology investments with risk

Advanced manufacturing system technologies are difficult to evaluate using ordinary financial measures owing to the presence of risk and significant intangible factors. In this paper a methodology is proposed that quantifies financial, strategic, and tactical attributes of each alternative. Attribute values are denned for each period of project life. Both project life and attributes are treated as random variables. Attributes are discounted to the current time and the multiple objectives are combined by composite programming. The distribution of the combined objective is estimated. The procedure is demonstrated on an actual case study.     

Trypsin inhibitor activity of soybean as affected by genotype and fertilisation

The presence of protease inhibitors in soybean prohibits the utilisation of the raw beans for food and feed. However, little information is available about environmental influences and the effects of nitrogen and sulphur supply on the antinutritional constituents of soybean. As these factors may influence protease inhibitors, soybean genotypes segregated according to the presence or absence of the Kunitz trypsin inhibitor have been evaluated for trypsin inhibitor activity (TIA) in field trials. TIA was affected significantly by environment (geographical location), fertilisation treatment and genotype. Environmental means of TIA were between 69.5 and 104.8 mg g^?1. Nitrogen application, which caused an increase in seed protein content, resulted in a reduction in TIA by about 15% as compared with the control. Remarkably, simultaneous application of nitrogen and sulphur in the form of ammonium sulphate had a similar reductive effect on TIA to that of nitrogen application alone, although soybean protease inhibitors are rich in sulphur amino acids. Significant genetic variation in TIA was found both within the genotype class with the Kunitz inhibitor present as well as within the class lacking this inhibitor. The results suggest that TIA of soybean may be modified considerably by genetic improvement and appropriate agronomic management. Copyright © 2003 Society of Chemical Industry     

A survey on the occurrence of Fusarium mycotoxins in Bavarian cereals from the 1987 harvest

Summary Bavarian cereals and wheat flour from the 1987 harvest were analysed for nivalenol (NIV) and deoxynivalenol (DON) using high performance liquid chromatography (HPLC) and for T-2 toxin and zearalenone (ZEA) by enzyme-linked-immunosorbent as say (ELISA). The study included 190 field samples of wheat, barley, rye and oat with visibly damaged ears, 45 samples of wheat intended for feed production and two series of wheat flour (type 550) and whole wheat flour collected in October 1987 and June 1988. The field samples examined showed a high DON contamination of wheat (87%) with an average of 3.96 mg/kg and a maximum of 43.8 mg/kg. Mean levels between 0.33 mg/kg and 0.27 mg/kg DON could be detected in barley, rye and oat. Of the wheat samples, 58% contained ZEA with a maximum of 1.560 mg/kg. The highest levels of ZEA were detected in samples which also showed high concentrations of DON. The NIV and T-2 toxin levels were comparatively low. Thirty percent of the samples showed NIV concentrations between 0.04 mg/kg and 0.29 mg/kg and 38% contained between 0.005 and 0.60 mg/kg of T-2. In the wheat samples for feed production, only DON was detected with an average of 0.190 mg/kg and a maximum of 0.75 mg/kg. The highest DON levels (0.58 mg/kg) from October 1987 were found in the wheat flour samples which were lower than the highest DON concentration (3.24 mg/kg) detected in the samples collected during June 1988. This fact was probably due to a substantial amount of non-contaminated wheat from 1986. The toxin concentrations in the whole wheat flour were not higher than in the type 550 flour. The regional distribution of the mean DON concentrations showed the highest levels in Middle and Lower-Franconia.

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Vorkommen von Fusarium Mykotoxinen in bayerischem Getreide der Ernte 1987

Zusammenfassung Cerealien und Weizenmehle der bayerischen Ernte 1987 wurden mittels hochauflösender Flüssigchromatographie (HPLC) auf Nivalenol (NIV) und Deoxynivalenol (DON) Bowie mit Enzymimmunoassay auf T-2 Toxin und Zearalenon (ZEA) analysiert. Die Untersuchungen umfaßten 190 Feldproben von Weizen, Gerste, Roggen und Hafer, die alle optisch erkennbaren Fusarienbefall aufwiesen, 45 Futterweizenproben Bowie zwei Probenserien von Weizenmehlen der Type 550 und Vollkornweizenmehlen, die im October 1987 und im Juni 1988 gezogenwurden. — Die Untersuchungen der Feldproben ergaben eine hohe DON-Kontamination des Weizens (87%) mit einem durchschnittlichen Gehalt von 3,96 mg/kg und einem Maximalgehalt von 43,8 mg/kg. In Gerste, Roggen und Hafer konnten durchschnittlich zwischen 0,33 mg/kg und 0,27 mg/kg DON-nachgewiesen werden. 58% der Winterweizenproben wiesen Zearalenon mit einem Maximalgehalt von 1,56 mg/kg auf. Die höchsten ZEA-Werte wurden in Proben ermittelt, die gleichzeitig einen hohen DON-Gehalt aufwiesen. Die Konzentrationen von NIV und T-2 Toxin waren vergleichsweise niedrig. 30% der Proben hatten NIV-Gehalte zwischen 0,04 mg/kg und 0,29 mg/kg und 38% enthielten T-2 Toxin zwischen 0,005 mg/kg und 0,06 mg/kg. In den Futterweizenproben konnte DON als einziges Toxin mit einem Gehalt von durchschnittlich 0,19 mg/kg und maximal 0,75 mg/kg festgestellt werden. Die Weizenmehle, die im October 1987 gezogen wurden, wiesen maximal 0,58 mg/kg DON auf. Die Gehalte lagen damit medriger als die der Mehlproben vom Juni 1988, die maximal 3,24 mg/kg und durchschnittlich 0,26 mg/kg DON enthielten. Dieser Sachverhalt könnte auf Anteile von nicht kontaminiertem Weizen der Ernte 86 an den im October gezogenen Mehlproben zurückgeführt werden. Die Toxingehalte der Vollkornmehle waren nicht höher als die der Weizenmehle der Type 550. Die höchsten Durchschnittsgehalte von DON wurden in Mittel- und Unterfranken festgestellt.

     

Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s_0.5,benz = 61.2 µM), k_cat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s_0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s_0.5,benz = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.     

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.     

An ionene with spirane structure (spiroionene)

The secondary diamine 1,3,5,7-tetrahydro[1,2c:4,5c'] benzodipyrrole (3) and 1,2,4,5-tetrabromomethylbenzene (1) form a polymeric ionene with spirane structure through a repetitive alkylation reaction. The structure of the product could be proven by¹³C-NMR spectroscopy by comparison with suitable reference compounds. Solutions in aqueous methanol exhibit a typical polyelectrolyte effect. Variation of the counterions produces sufficient solubility in organic solvents. From the crystal structure of a similar model compound one can conclude that the synthesized polymer has a rod-like shape.     

High-Throughput Experimentation in Oxidation Catalysis

High-throughput experimentation in catalysis comprises the following components: (i) automated high-throughput synthesis, (ii) testing in Stage I and Stage II, for which to some extent novel assays are necessary, (iii) data handling and experimental design tools, and (iv) robotics. This contribution covers these topics, using examples from the research of the authors, but also from the literature, in order to illustrate the problems and opportunities associated with high-throughput experimentation in catalysis, focusing particularly on heterogeneous catalysis.