A fungal enzyme with the ability of aflatoxin B₁ conversion: purification and ESI-MS/MS identification

Armillariella tabescens, a Chinese edible and medicinal fungus, whose multienzyme exist ability of AFB₁-converting, and ADTZ (aflatoxin-detoxizyme) had previously purified from the A. tabescens multienzyme monitored by AFB₁ conversion_. However, the enzyme now confirmed an oxidase and renamed aflatoxin-oxidase (AFO). In this paper, AFO was purified by an economical and practical three-step procedure monitored by AFB₁ conversion. And ESI-MS/MS analysis was done for identification of AFO. The following database searching (Protein Blast on NCBI) results did not show any homologous oxidase protein, which implied that AFO was mostly a new oxidase differing from other reported aflatoxin-converting enzymes such as fungal laccase and horse radish peroxidase. HPTLC analysis of the purified AFO activity suggested that the enzyme reacted at the bisfuran ring of AFB₁ which was the key toxic structure. Therefore, all these investigations implied a new choice for biodegradation of aflatoxin in foods and feeds with the practical application of AFO.     

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 μg/mL corresponding to 50 μM, 24–48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3β), and with degradation of Mcl-1. With α-tocopherol (400 μM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3β dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3β/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.     

A novel method for analyzing phosphoproteins using SELDI-TOF MS in combination with a series of recombinant proteins

A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS.     

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI‐TOF‐MS

The steady‐state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI‐TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP‐Hex, UDP‐HexNAc). By switching to a ¹³C₆ glucose containing feed media during constant operation at 20 × 10⁶ cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the ¹³C₆ glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time (1 day) and characteristic time for glucose uptake (0.33 days), representative of the bioreactor dynamics, delayed the consumption of ¹³C₆ glucose in the bioreactor and thus the intracellular ¹³C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630–1639, 2017     

菊花不同花期及花序不同部位香气成分和挥发研究

以切花菊品种‘神马’为试材,采用顶空-固相微萃取和气相色谱-质谱联用(GC/MS)技术,分别测定菊花不同花期及花序不同部位的香气成分种类和含量,并利用生物显微镜观察花瓣的表皮细胞和横切面组织细胞的结构特征。结果表明:(1)菊花花蕾期共检测到香气成分24种,始花期31种,盛花期43种,终花期22种;随着花朵的开放和凋谢,酮类、萜烯类和醇类化合物的含量呈先上升后下降的趋势,在盛花期含量达到最高,而酯类、醛类和杂环类化合物则呈持续下降的趋势。(2)盛花期,在舌状花中共检测到香气成分种类31种,在管状花中共检测到50种;舌状花对菊花香气的贡献比管状花大;菊花舌状花由内轮向外轮香气成分种类变化不大,但是同类香气成分含量的变化出现由内轮向外轮逐渐减少的趋势。(3)异环柠檬醛、桉叶醇、α-蒎烯、β-金合欢烯和石竹烯等化合物可能为菊花的主要特征香气成分。(4)显微观察结果表明:舌状花的香气可能是通过表皮细胞间隙释放的,上表皮是菊花释放香气的主要部位。     

Peptidome analysis reveals the involvement of endogenous peptides in mouse pancreatic dysfunction with aging

Type 2 diabetes (T2D) is a glucose regulation disorder that has significantly enhanced mortality and the global disease burden. The prevalence of T2D has increased worldwide and is higher in the elderly. The function of pancreatic islets decreases with age, which is one important reason for the occurrence of diabetes in the elderly. Recently, peptidome analysis has attracted attention. However, the role of age-related peptides in pancreatic dysfunction has not been investigated extensively. Here, we conducted a comparison of endogenous peptides between pancreas from adult and aging mice by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 2,089 peptides originating from 1,280 protein precursors were identified, of which 232 were upregulated and 183 were downregulated in the aging mice (fold change ≥ 2 and p < 0.05), suggesting that the expression of pancreatic peptides in mice varied with age. The molecular weight of most peptides was <3.0 kDa, and the isoelectric point distribution had a bimodal characteristic. Further analysis of cleavage site patterns indicated that proteases cleaved pancreatic proteins according to their rules. Moreover, Gene Ontology and pathway analyses showed that the differentially expressed peptides potentially had specific effects on pancreatic dysfunction. Some differential peptides were located within the domains of precursor proteins that were closely associated with the development of diabetes. We believe that our research may advance the current understanding of pancreas-derived peptides and that certain peptides may be involved in the etiology of diabetes.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Exploring natural variation of Pinus pinaster Aiton using metabolomics: Is it possible to identify the region of origin of a pine from its metabolites?

Natural variation of the metabolome of Pinus pinaster was studied to improve understanding of its role in the adaptation process and phenotypic diversity. The metabolomes of needles and the apical and basal section of buds were analysed in ten provenances of P. pinaster, selected from France, Spain and Morocco, grown in a common garden for 5 years. The employment of complementary mass spectrometry techniques (GC‐MS and LC‐Orbitrap‐MS) together with bioinformatics tools allowed the reliable quantification of 2403 molecular masses. The analysis of the metabolome showed that differences were maintained across provenances and that the metabolites characteristic of each organ are mainly related to amino acid metabolism, while provenances were distinguishable essentially through secondary metabolism when organs were analysed independently. Integrative analyses of metabolome, environmental and growth data provided a comprehensive picture of adaptation plasticity in conifers. These analyses defined two major groups of plants, distinguished by secondary metabolism: that is, either Atlantic or Mediterranean provenance. Needles were the most sensitive organ, where strong correlations were found between flavonoids and the water regime of the geographic origin of the provenance. The data obtained point to genome specialization aimed at maximizing the drought stress resistance of trees depending on their origin.     

Modification of quercetin with l-cysteine by horseradish peroxidase

Horseradish peroxidase is a well-known member of the peroxidase family that catalyzes oxidation of flavonoids and phenolic substrates to free phenoxyl or semiquinone radicals. Aim of this study was to investigate in vitro oxidation of quercetin by horseradish peroxidase in the presence of l-cysteine as nucleophilic agent, and its influence on previously formed semiquinone- and quinone-type metabolites. The obtained results showed that in the reaction without l-cysteine several products were present, such as quercetin quinone methide, phloroglucinol carboxylic acid, protocatechuic acid, as well as quercetin heterodimer and derivates of quercetin heterodimer. On the other hand, in the presence of l-cysteine only three products were obtained, quercetin quinone methide and two new isomeric mono-cysteine derivatives of quercetin with mass exp. m/z 420.04?±?0.1 [quercetin?+?cysteine–H]^– (theor. m/z 420.0389 [quercetin?+?cysteine–H]^–).     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.