Serine hydrolases in differentiating Rhodomicrobium vannielii swarmer cells

³H-diisopropylfluorophosphate (DFP) was used to specifically label serine hydrolases in differentiating Rhodomicrobium vannielii swarmer cells. Fluorography of SDS-polyacrylamide gels revealed several changes in the pattern of ³H-DFP-labelled polypeptides, including the appearance of a new species and apparent increases in the amounts of others. Most of these changes occurred late in differentiation and may be associated with cell division events.Abbreviations DFP diisopropylfluorophosphate - SDS sodium dodecyl sulphate     

Partial Characterization of the Cuticle Surface of Meloidogyne javanica Females

Negative charges on the outer cuticular surface of Meloidogyne javanica females were visualized with electron microscope labelling techniques. Evidence is presented that the electronegative charge is not borne on neuraminic acid. Ruthenium red staining indicated acid mucopolysaccharides on the outer surface. A surface coat, or glycocalyx, external to the outer cuticle membrane was demonstrated.     

Metabolic flux analysis in plants: coping with complexity

Theory and experience in metabolic engineering both show that metabolism operates at the network level. In plants, this complexity is compounded by a high degree of compartmentation and the synthesis of a very wide array of secondary metabolic products. A further challenge to understanding and predicting plant metabolic function is posed by our ignorance about the structure of metabolic networks even in well-studied systems. Metabolic flux analysis (MFA) provides tools to measure and model the functioning of metabolism, and is making significant contributions to coping with their complexity.
This review gives an overview of different MFA approaches, the measurements required to implement them and the information they yield. The application of MFA methods to plant systems is then illustrated by several examples from the recent literature. Next, the challenges that plant metabolism poses for MFA are discussed together with ways that these can be addressed. Lastly, new developments in MFA are described that can be expected to improve the range and reliability of plant MFA in the coming years.     

Carbon labelling influences on consumers’ behaviour: A system dynamics approach

This paper proposes a system dynamics approach to model consumers’ responses to carbon labelled products by showing how the number of consumers is varied visually. Three categories of the consumers are defined, i.e., potential, ordinary and loyal consumers, to reflect their different preferences to the carbon labelled products. Two case scenarios are presented to demonstrate the application of the proposed approach, in which the consumers’ preferences to the carbon labelled and non-labelled milk, as well as the carbon labelled milk with different fat contents are simulated. The result shows that both of the two scenarios have the similar variation in the number of the defined consumers, and especially the number of loyal consumer accounts for the largest proportion. In addition, public awareness, education level, critical premium and perceived consumer effectiveness are identified as key factors to have an impact on the purchasing behaviour. The limitations of this approach are then discussed, laying a foundation for further work.     

不同转移潜能膀胱癌细胞糖组相对定量分析

膀胱癌是发生在膀胱黏膜组织上的一种恶性肿瘤,是泌尿系统中最常见的恶性肿瘤,早期(非肌层浸润型膀胱癌)阶段的诊断和治疗是降低膀胱癌死亡率的最有效方式.肿瘤的发生过程与糖链表达的改变有着密切的关系,而定量分析膀胱癌发生过程中糖链的表达变化尚未有研究.本研究以2株人膀胱正常上皮细胞系(HCV29、HUCV1),1株非肌层浸润性膀胱癌细胞系(KK47),和3株浸润性膀胱癌细胞系(YTS1、J82、T24)为研究材料,应用本室建立的利用乙酰肼修饰糖链唾液酸,以及[12C6]-和[13C6]-苯胺同位素修饰糖链还原性末端技术,然后利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS),进行膀胱上皮细胞不同病理状态的糖组相对定量分析.从6株细胞中共鉴定出52种N-连接糖链结构,并定量分析了不同类型的糖链在不同细胞中的分布差异,发现唾液酸化、岩藻糖化的N-连接糖链在膀胱癌肿瘤细胞恶化过程中呈现显著升高的趋势,同时平分型糖链和高甘露糖型N-连接糖链也呈表达升高趋势,说明这些糖链结构的表达变化与膀胱癌发生关系密切,从而有助于进一步阐明膀胱癌发生过程中糖链相关的分子机理.     

Kinetics and localisation of haemin-induced lipoprotein oxidation

Abstract

Haemin (iron (III)-protoporphyrin IX) is a degradation product of haemoglobin in circulating erythrocytes. Haemin may play a key oxidising agent for lipoprotein oxidation in patients with haemolytic anaemia. In this study, kinetic changes in chemical composition and target sites of haemin-induced LDL and HDL oxidation were investigated. Haemin initially induced the loss of α-tocopherol, followed by accumulation of lipid hydroperoxide (LP) and alteration of core lipid fluidity. The absence of LP in HDL was explained by the antioxidant activity of PON in addition to α-tocopherol. The target site of haemin was evaluated by ESR spin labelling with 5- and 16-doxyl steric acids. In the presence of t-BuOOH and haemin, ESR signal decay of the doxyl moiety demonstrated the initiation phase and the propagation phase of lipid peroxidation. The results of the lag time and the rate of signal decay indicated that haemin is located near the 16th carbon atom of the fatty acid chain in the phospholipid layer. The analyses of motion parameters, order parameter (S) of 5-DS and rotational correlation time (τ) of 16-DS, supported the observation that the lipid properties changed near the hydrophobic region rather than at the surface region of lipoproteins. Moreover, ESR spin labelling demonstrated that haemin molecules but not iron ions caused lipoprotein oxidation. In conclusion, haemin is a potent inducer of lipoprotein oxidation, and the target site for this oxidation is near the hydrophobic core of the lipoprotein leading to the loss of antioxidant activities and changes in lipid composition and physical properties.     

Interactive effects of drought and N fertilization on the spatial distribution of methane assimilation in grassland soils

Soil methanotrophic bacteria constitute the only globally relevant biological sink for atmospheric methane (CH₄). Nitrogen (N) fertilizers as well as soil moisture regime affect the activity of these organisms, but the mechanisms involved are not well understood to date. In particular, virtually nothing is known about the spatial distribution of soil methanotrophs within soil structure and how this regulates CH₄ fluxes at the ecosystem scale. We studied the spatial distribution of CH₄ assimilation and its response to a factorial drought × N fertilizer treatment in a 3‐year experiment replicated in two grasslands differing in management intensity. Intact soil cores were labelled with ¹⁴CH₄ and methanotrophic activity mapped at a resolution of ～100 μm using an autoradiographic technique. Under drought, the main zone of CH₄ assimilation shifted down the soil profile. Ammonium nitrate (NH₄NO₃) and cattle urine reduced CH₄ assimilation in the top soil, but only when applied under drought, presumably because NH₄⁺ from fertilizers was not removed by plant uptake and nitrification under these conditions. Ecosystem‐level CH₄ fluxes measured in the field did show no or only very small inhibitory effects, suggesting that deeper soil layers fully compensated for the reduction in top soil CH₄ assimilation. Our results indicate that the ecosystem‐level CH₄ sink cannot be inferred from measurements of soil samples that do not reflect the spatial organization of soils (e.g. stratification of organisms, processes, and mechanisms). The autoradiographic technique we have developed is suited to study methanotrophic activity in a relevant spatial context and does not rely on the genetic identity of the soil bacterial communities involved, thus ideally complementing DNA‐based approaches.     

Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes

Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[²H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D^−/− mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 μmol/lung/h) and SP-D^−/− (0.69 μmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D^−/− mouse was due to impaired catabolism, with a rate of accumulation of 0.057 μmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D^−/− mice before significant PC acyl remodelling occurs.     

Photo-oxygenation of α-Ionone

Photo-oxygenation of α-ionone was studied to clarify the relationship between the maturity of aroma and photo-oxygenative change of α-ionone. α-Ionone was converted to oxygenated derivatives which were identified as 2,3-epoxy-β-ionone, 3,4-epoxy-α-ionone, 4-keto-β-ionone (trans- and cis-form), 5-keto-α-ionone and 3,4-dihydroxy-α-ionone.     

Quantitative protein analysis using 13C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry

We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacetanilide (IAA, 1) and ¹³C₇-labeled iodoacetanilide (¹³C₇-IAA, 2), or N-ethylmaleimide (NEM, 3) and d₅-labeled N-ethylmaleimide (d₅-NEM, 4), followed by mass spectrometric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research.