Utilization of amino acids in growing kidney proximal tubule cell cultures

The growth of rat kidney proximal tubule cells was monitored continuously by the cellular incorporation of [methyl-(14)C] thymidine using scintillating microplates. The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine. Leibovitz L-15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids. Kidney proximal tubule cells had a greater potential to process amino acids than BHK-21 cells. The utilization of amino acids by proximal tubule cells differed from that of other organs. The amino acids could be classified into three classes. Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine). The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine).     

不同年龄大鼠尿液代谢组学研究

旨在探究健康大鼠的尿液代谢物在生长、发育和衰老过程中的年龄相关性变化.采集大鼠出生后3周、5周、7周、9周、12周、56周和111周共7个年龄点的尿液样本,利用GC/TOF-MS平台进行代谢组学检测.结果显示,所有尿液样本在主成分分析与偏最小二乘法判别分析中出现组内聚类和组间分离.氨基酸类、有机酸类和碳水化合物类代谢物...     

不同碳源和生长因子条件下粒毛盘菌代谢产物初步研究

采用GC-MS法对一种粒毛盘菌(Lachnum sp.)在不同碳源、生长因子条件下发酵代谢产物的挥发性成分组成与差异进行分析。结果显示,不同碳源和生长因子条件下产生的代谢产物不同,主要包括有机酸、胺类、烷烃类、酯类、醇类、吡咯等物质。分别以20 g/L的葡萄糖、蔗糖、淀粉为碳源的发酵液中检测到的挥发性代谢产物为7、7、10种;添加1 mg/L的V_C、V_(B1)、甘氨酸、色氨酸作为不同生长因子的发酵液中检测到的挥发性代谢产物分别为6、7、7、12种。结果显示粒毛盘菌YM406发酵代谢产物具有丰富的多样性,并且在不同的培养条件下产生的代谢产物存在一定的差异。     

Effect of secondary metabolites associated with anaerobic soil conditions on ion fluxes and electrophysiology in barley roots

The effects of secondary metabolites produced by waterlogged soils on net K(+), H(+), and Ca(2+) fluxes were studied in the mature zone of roots of two barley (Hordeum vulgare) cultivars contrasting in their waterlogging (WL) tolerance using the noninvasive microelectrode ion flux measuring technique. In WL-sensitive variety 'Naso Nijo', all three lower monocarboxylic acids (formic, acetic, and propionic acids) and three phenolic acids (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic acids) caused a substantial shift toward steady K(+) efflux, accompanied by an immediate net influx of H(+). Detrimental effects of secondary metabolites on K(+) homeostasis in root cells were absent in WL-tolerant 'TX' variety. Root treatment with Mn(2+) caused only a temporary K(+) loss that returned to the initial level 10 min after treatment. Phenolic acids slightly increased Ca(2+) influx immediately after treatment, while other metabolites tested resulted in transient Ca(2+) efflux from the root. In the long-term (24 h) treatment, all metabolites tested significantly reduced K(+) uptake and the adverse effects of phenolic acids were smaller than for monocarboxylic acids and Mn(2+). Treatment with monocarboxylic acids for 24 h shifted H(+) from net efflux to net influx, while all three phenolic acids did not cause significant effects compared with the control. Based on results of pharmacological experiments and membrane potential measurements, a model explaining the effects of secondary metabolites on membrane transport activity is proposed. We also suggest that plant tolerance to these secondary metabolites could be considered a useful trait in breeding programs.     

Metabolite production and growth efficiency

The capacity to sustain the large fluxes of carbon and energy required for rapid metabolite production appears to be inversely related to the growth efficiency of micro-organisms. From an overall energetic point of view three main classes of metabolite may be distinguished. These are not discrete categories, as the energetics of biosynthesis will depend on the precise biochemical pathways used and the nature of the starting feed stock(s). (1) for metabolites like exopolysaccharides both the oxidation state and the specific rate of production appear to be inversely related to the growth efficiency of the producing organism. Maximum rates of production are favored when carbon and energy flux are integrated, and alteration of this balance may negatively effect production rates. (2) The production of metabolites like organic acids and some secondary metabolites results in the net production of reducing equivalents and/or ATP. It is thought that the capacity of the organism to dissipate this product associated energy limits its capacity for rapid production. (3) For metabolites like biosurfactants and certain secondary metabolites that are composed of moieties of significantly different oxidation states production from a single carbon source is unfavorable and considerable improvements in specific production rate and final broth concentration may be achieved if mixed carbon sources are used. By careful selection of production organism and starting feedstock(s) it may be possible to tailor the production, such that the adverse physiological consequences of metabolite overproduction on the production organism are minimized.     

The aldehydic metabolites of linoleic acid are cytotoxic against human breast cancer cells

The cytotoxic effect of aldehydic metabolites of linoleic acid, 13-oxo-tridecadienoic acids, on MCF-7 human breast cancer cells was investigated. The metabolites inhibited the growth of the cancer cells and the effect was dependent on both time of exposure and concentration of the metabolites; 50% growth inhibition occurred at approximately 55 and 33 microM, after 3- and 5-day incubations, respectively. The metabolites had greater cytotoxicity than parent linoleic acid or other polyunsaturated fatty acids tested. The antiproliferative effect was partially reversed by 10 microM of dithiothreitol suggesting that attack on thiol groups in cancer cells by highly reactive alpha, beta-unsaturated carbonyl moiety in the metabolites was responsible for the cytotoxic actions.     

Metabolic responses of wheat roots to alkaline stress

Aims The aim of this study was to investigate the effects of alkaline stress on primary, secondary metabolites and metabolic pathways in the roots of wheat (Triticum aestivum). The results were used to evaluate the physiological adaptive mechanisms by which wheat tolerated alkali stress.Methods A pot experiment was carried out in the greenhouse. For each plastic pot, five wheat seeds were planted. After germination, seedlings were allowed to grow under controlled water and nutrient conditions for two months, then seedlings were exposed to alkaline stress (NaHCO₃-Na₂CO₃) for 12 days. The relative growth rate (RGR), absolute water content (AWC), metal elements, free cations and metabolites were measured.Important findings The alkaline stress caused the reduction of RGR and AWC. Alkaline stress caused a rapid increase of Na content with the concurrent decrease in K and Cl content, resulting in inhibited metal element accumulation and an ionic imbalance. In the present study, alkaline stress strongly enhanced Ca accumulation in wheat roots, suggesting that an increased Ca concentration can immediately trigger the salt overly sensitive (SOS)-Na exclusion system and reduce Na-associated injuries. Also, 70 metabolites, including organic acids, amino acids, sugars/polyols and others, behaved differently in the alkaline stress treatments according to a GC-MS analysis. The metabolic profiles of wheat were closely associated with alkaline-stress conditions. Alkaline stress caused the accumulation of organic acids, accompanied by the depletion of sugars/polyols and amino acids. Organic acids could play a central role in the regulation of intracellular pH by accumulating vacuoles to neutralize excess cations. Glycolysis and amino acid synthesis in roots were inhibited under salt stress while prolonged alkaline stress led to a progressive tricarboxylic acid (TCA) cycle. The severe negative effects of alkaline stress on sugar synthesis and storage may reflect the toxic levels of Na⁺ accumulating in plant cells in a high-pH environment, implying that the reactive oxygen species detoxification capacity was diminished by the high pH. A lack of NO₃^- in wheat roots can decrease synthase enzyme activities, limiting the synthesis of amino acids. Under salt stress, the TCA cycle and organic acid accumulation increased, but glycolysis and amino acid synthesis were inhibited in roots. Thus, energy levels and high concentrations of organic acids may be the key adaptive mechanisms by which wheat seedlings maintain their intracellular ion balance under alkaline stress.     

Growth metabolism of wheat under drought stress at the jointing-booting stage

AimsThe aim of this study was to investigate the effects of drought stress on primary, secondary metabolites and metabolic pathways in the leaves of wheat, these parameters were evaluated to determine the physiological adaptive mechanisms by which wheat tolerates drought stress at the jointing-booting stage.MethodsA pot experiment was carried out in rain-proof shelter. The relative growth rate, photosynthetic characteristics and metabolism seedlings exposed to stresses lasting 12 days at jointing-booting stage were measured.Important findings The results displayed that the photosynthesis decreased under drought stress, causing the decreases of relative growth rate and dry matter mass. Profiles of 64 key metabolites produced by wheat including organic acids, amino acids, carbohydrates, purine, etc. were examined, 29 of them were changed significantly under drought stress. Principal component analysis (PCA) showed that 64% variations can be explained by the two principal components. One-way ANOVA analysis results revealed that long term drought stress decreased malic acid, citric acid and aconitic acid significantly, indicating inhibited tricarboxylic acid cycle. We further found that prolonged drought stress led to accumulation of progressive amino acids (proline, serine, valine) and carbohydrates (myo-inositol, fructose, clucose) in wheat leaves and depletion of transamination products (asparagine, glutamine, γ-aminobutyric acid). These results imply wheat may enhance its drought tolerance mainly by increasing amino acid biosynthesis and glycolysis under water-deficit conditions. Our findings suggest that drought condition altered metabolic networks including transamination, the tricarboxylic cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, and the metabolisms of choline, pyrimidine and purine. This study provides new insights into the metabolic adaptation of wheat to drought stress and important information for developing drought-tolerant wheat cultivars.     

拔节孕穗期小麦干旱胁迫下生长代谢变化规律

采用盆栽试验模拟干旱胁迫(土壤相对含水量40%-45%)在小麦(Triticum aestivum)拔节孕穗期胁迫12天, 测定其生长速率、光合特征及关键代谢产物含量, 以探讨干旱胁迫对拔节孕穗期小麦叶片初生及次生代谢产物的影响及其涉及的代谢途径, 讨论小麦生长代谢变化规律及应答机制。研究表明: 干旱胁迫使小麦叶片气孔受限制导致光合速率下降; 使叶绿素含量下降直接影响光系统II活性, 最终导致生长率降低。检测出的初级代谢产物组包括有机酸、氨基酸、碳水化合物、嘧啶和嘌呤等64个代谢产物, 其中29个代谢产物在干旱胁迫下发生明显的变化。主成分分析(PCA)结果显示全部样本均分布在95%的置信区间内, 两个主成分得分为64%。单因素方差分析结果表明, 干旱胁迫导致苹果酸、柠檬酸、乌头酸等参与三羧酸(TCA)循环的代谢产物消耗明显, 且引起大部分氨基酸(如脯氨酸、丝氨酸、缬氨酸)和碳水化合物(肌醇、果糖、葡萄糖)大量积累的同时转氨基代谢(天冬酰胺、谷氨酰胺和γ氨基丁酸)产物消耗, 研究证明干旱胁迫明显地促进小麦叶片的糖酵解和氨基酸合成途径, 但抑制了TCA循环和转氨基反应, 加速氨基酸代谢网络向脯氨酸合成转变过程。这些结果表明干旱胁迫引起了转氨基反应、TCA循环、糖酵解/糖异生、谷氨酸介导的脯氨酸合成, 以及嘧啶和嘌呤等代谢网络系统广泛的变化, 说明小麦在合成大量的氨基酸和碳水化合物类物质的同时也消耗了大量的能量, 暗示了糖异生到脯氨酸合成的转变。     

常见肠道菌群代谢产物作为疾病诊断的指针的研究进展

人类肠道菌群能够产生多种代谢产物或与人体相互作用产生肠道菌群-宿主共代谢物,显著影响人体各大系统的生理功能。当人体健康状态以及肠道菌群发生变化时,肠道代谢物的种类和含量也会相应受到影响,因此肠道菌群代谢产物具有作为疾病诊断指针的巨大潜力。本文总结了常见的几类肠道微生物代谢产物,包括糖类、胆碱代谢物、脂质、氨基酸与肽类、维生素、胆汁酸、短链脂肪酸、酚、苯甲酰基和苯基衍生物等,及其在不同疾病状态下的作用机理,以期更好地理解肠道菌群、代谢产物和疾病之间的相关性,为疾病的预防、诊断和治疗提供新的靶点。     

冠突曲霉 veA基因缺失型与野生型的差异代谢物研究

为了鉴定冠突曲霉 veA基因缺失型与野生型的差异代谢物,寻找与 veA 基因产孢相关的代谢物,采用气相色谱-质谱联用（GC-MS）的代谢组学技术,分别收集2个菌株培养48h的菌丝体,经液氮研磨、超声萃取、三甲基氯硅烷衍生化后上机检测,将GC-MS检测的数据进行前处理和代谢物注释,并用统计学相关软件进行差异物筛选和相关性分析。结果显示,共鉴定99种代谢物,其中差异代谢物41种,野生型菌株中表达上调的显著差异代谢物有20种, veA缺失型菌株中表达上调的显著差异代谢物有21种,并预测差异代谢物的相关性,发现与623种代谢物产生相关性,其中313种呈正相关,310种呈负相关。筛选出来的差异代谢物涉及有机酸、氨基酸、碳水化合物、醇类、脂肪酸类等多种代谢物质,其中有机酸和氨基酸类代谢物占主导地位。本研究结果为进一步研究冠突曲霉代谢物与产孢的关联提供了一定的理论基础。     

Metabolomics Analysis of Metabolites in <i>Forsythia suspense</i> Fruit Using UPLC/ESI-Q TRAP-MS/MS

Forsythiae Fructus, the fruit of Forsythia suspense is a traditional Chinese hebal medicine that has the antiviral and antioxidant effects in China. Modern analytical chemistry studies showed that the extracts of Forsythiae Fructus contain many bioactive components, such as flavonoids, lignans, phenolic acids, and terpenoids, which can be used to anti-inflammatory and treat toxicity, tonsillitis, ulcers, pharyngitis and acute nephritis. In order to study the types and quantities of metabolites in Forsythiae Fructus, we isolated, identified and analysed metabolites between two varieties of Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS. The results showed that a total of 407 metabolites were identified in Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS, including 21 terpenoids, 68 phenolic acids, 63 flavonoids, 43 amino acids and derivatives, 22 alkaloids, 55 lipids, 24 lignans and coumarins, 31 nucleotides and derivatives, 29 organic acids, and 51 other metabolites. Among, lignans and coumarins, terpenoids, organic acids, lipids, and phenolic acids were rich in Forsythiae Fructus, which accounted for more than 60% of the total metabolite content. Differential metabolite analysis revealed that 80 metabolites differed significantly between the two types of Forsythiae Fructus. Our results greatly enrich the Forsythiae Fructus phytochemical composition database and provide valuable information for further study of the metabolites of Forsythiae Fructus.     

不饱和脂肪酸参与调控金针菇菌柄伸长的潜在分析

金针菇Flammulina filiformis菌柄是产量构成的重要组成部分,也是评价质量的重要指标。菌盖下方0.6-1.5cm是菌柄的伸长区,其他区段不伸长。为了研究菌柄伸长的调控机制,我们采用液相色谱-串联质谱联用（LC-MS/MS）技术对金针菇菌柄的伸长区、过渡区和非伸长区进行检测,结合层次聚类分析（HCA）、t检验等统计学方法对差异代谢物进行研究,并通过KEGG分析代谢物富集情况。结果显示,共有69种代谢物在伸长区、过渡区和非伸长区呈现梯度差异,其中52种代谢物含量为梯度性降低,17种代谢物为梯度性增加。这些差异代谢物主要归属于脂肪酸、氨基酸、核苷酸、生物碱等,其中脂肪酸及其衍生物占主导地位,尤其是油酸、花生四烯酸和肾上腺酸含量在菌柄伸长区显著高于其他区段。根据金针菇菌柄的代谢物积累特点,推测脂肪酸及其衍生物可能参与调控金针菇菌柄伸长。     

两种光照处理下苦荞芽菜的代谢物分析

以黑暗和光照处理下苦荞（Fagopyrum tataricum（L.）Gaertner）品种‘晋荞2号’种子萌发所得的芽菜为材料,利用气相色谱-质谱联用分析技术,对两种处理下苦荞芽菜代谢产物的差异进行分析。结果显示,两种光照处理下苦荞芽菜的代谢产物差异明显,在芽菜中共检测出383种代谢产物,其中不同光照处理造成了137种代谢产物的显著差异,他们主要集中在氨基酸及其衍生物、糖类及其衍生物、芳香族化合物和脂肪酸等。除氨基酸及其衍生物外,其余绝大多数差异代谢物的含量在光照组芽菜中较高;KEGG代谢通路分析结果表明,差异代谢物富集于136条物质代谢通路。说明光照能够促进苦荞芽菜多数营养代谢物的生成和积累,营养组分较暗培养更为丰富,营养价值更高。     

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30° C but no detectable diauxic lag at 5° C.At 30° C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed unitl onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with respression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase.At 5° C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase.Thus, at low temperatures, psychrotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.     

Impact of Lactobacillus paracasei IMC502 in coculture with traditional starters on volatile and non-volatile metabolite profiles in yogurt

The impact of Lactobacillus paracasei IMC502 co-fermented with traditional starters on metabolites in yogurt was evaluated using metabolomic analysis. Forty-four volatile metabolites were determined using headspace solid phase microextraction and gas chromatography-mass spectrometry, including alcohols, esters, organic acids, carbonyl and sulfur compounds. Acetaldehyde, diacetyl, acetoin, acetone, butanoic and acetic acid were present in yogurts in higher intensity, which are the major volatile metabolites related to yogurt flavor. L. paracasei IMC502 did not affect the amounts of acetaldehyde, diacetyl and acetoin while promoted the formation of acetone and butanoic acid. A total of 196 non-volatile metabolites including nucleosides, amino acids, carbohydrates, lipids were analyzed using UPLC-Q-TOF-MS. Non-volatile metabolite profiles of these two types of yogurts were distinguished and 94 differential metabolites were screened using multivariate statistical analysis, which were mainly associated with the biosynthesis of secondary metabolites, amino acid metabolism and nucleotide metabolism. The impact of storage on metabolites was also investigated. The amounts of the majority of carbonyl compounds, organic acids and free amino acids increased, while those of acetaldehyde, diacetyl and lactose decreased during storage. This study provides insights into the metabolic mechanism of L. paracasei and represents a real advance in the study of the metabolites in yogurt.     

肠道微生物代谢物在肝硬化中的作用

肝硬化是由多因素引起的终末期肝病,也是导致慢性肝病患者病死的主要原因之一。随着微生物相关多组学的发展和应用,越来越多的研究发现,肠道微生物及其代谢产物如短链脂肪酸、胆汁酸和内毒素等在肝硬化等肝脏疾病中发挥主要作用,但这些肠道微生物代谢产物影响肝硬化发展的机制依然不明确。因此,根据近年来关于肠道微生物及其代谢产物与肝硬化关系的研究,本文重点介绍几种研究比较深入的微生物代谢物以及其影响肝硬化发展的潜在的作用机制,并简要介绍微生物代谢物治疗肝硬化的方法,以期为肝硬化的病理生理学机制、诊断和治疗提供新的见解。

     

Corticosteroid side-chain oxidations--I. Structural effects on the excreted isotope ratios of 4-14C- and 21-3H-labelled corticosteroid metabolites in rabbit urine

The metabolic fates of 4-14C- and 21-3H-labelled corticosteroids have been investigated in the rabbit by analysis of the normalized isotope ratios of neutral and acidic metabolites excreted in the urine. Isotope ratios of excreted radioactivity declined in the order cortisol (F) greater than corticosterone (B) greater than 11-desoxycortisol (S) greater than deoxycorticosterone (DOC). Steroid acids, isolated in alumina fraction C, represented 19.0, 15.0, 9.7 and 2.7% of the doses of DOC, B, S and F, respectively, and the isotope ratios declined in the order F greater than B greater than S greater than DOC. HPLC of steroid acid methyl ester derivatives indicated generally low isotope ratios for DOC and S steroid acids, consistent with complete side-chain oxidation to 20-oxo-21-oic acids and/or 17-carboxylic acids. Several B metabolite methyl esters peaks also exhibited low isotope ratios, but both B and F metabolites gave methyl esters that retained significant tritium consistent with the presence of 20-hydroxysteroid acids. The 21-hydroxy-steroid metabolite fractions had isotope ratios of F = S greater than B greater than DOC. HPLC showed that 20-oxo (tetrahydro) metabolites of B and F had reduced isotope ratios unlike the C-20 reduced (hexahydro) metabolites of DOC and S. It may be concluded that the metabolic fate of the corticoid side-chain in the rabbit is dependent on the steroid structure and may result in the excretion of both 20-oxo and 20-hydroxysteroid acids.     

Regulatory functions of bacterial exometabolites

This review deals with the issue of growth autoregulation and survival in bacterial cultures under starvation conditions. Based on our results and on published data, the conclusion has been drawn that low-molecular products of metabolism (carboxylic acids, amino acids, and other metabolites) perform regulatory functions. The same compounds also control the ecological relationship between microorganisms at the interspecific level, and affect their antagonistic activity. It is suggested that complexes of bacterial metabolites can be used for controlling the composition of various microbiocenosis, including those of humans.