Combination of ‘omics’ data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in Rats and to identify potential biomarkers

To gain novel insight into the molecular mechanisms underlying hydrazine-induced hepatotoxicity, mRNAs, proteins and endogenous metabolites were identified that were altered in rats treated with hydrazine compared with untreated controls. These changes were resolved in a combined genomics, proteomics and metabonomics study. Sprague–Dawley rats were assigned to three treatment groups with 10 animals per group and given a single oral dose of vehicle, 30 or 90 mg?kg^?1 hydrazine, respectively. RNA was extracted from rat liver 48 h post-dosing and transcribed into cDNA. The abundance of mRNA was investigated on cDNA microarrays containing 699 rat-specific genes involved in toxic responses. In addition, proteins from rat liver samples (48 and 120/168 h post-dosing) were resolved by two-dimensional differential gel electrophoresis and proteins with changed expression levels after hydrazine treatment were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting. To elucidate how regulation was reflected in biochemical pathways, endogenous metabolites were measured in serum samples collected 48 h post-dosing by 600-MHz ¹H-NMR. In summary, a single dose of hydrazine caused gene, protein and metabolite changes, which can be related to glucose metabolism, lipid metabolism and oxidative stress. These findings support known effects of hydrazine toxicity and provide potential new biomarkers of hydrazine-induced toxicity.     

Combination of 'omics' data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in Rats and to identify potential biomarkers

To gain novel insight into the molecular mechanisms underlying hydrazine-induced hepatotoxicity, mRNAs, proteins and endogenous metabolites were identified that were altered in rats treated with hydrazine compared with untreated controls. These changes were resolved in a combined genomics, proteomics and metabonomics study. Sprague-Dawley rats were assigned to three treatment groups with 10 animals per group and given a single oral dose of vehicle, 30 or 90 mg kg^-1 hydrazine, respectively. RNA was extracted from rat liver 48 h post-dosing and transcribed into cDNA. The abundance of mRNA was investigated on cDNA microarrays containing 699 rat-specific genes involved in toxic responses. In addition, proteins from rat liver samples (48 and 120/168 h post-dosing) were resolved by two-dimensional differential gel electrophoresis and proteins with changed expression levels after hydrazine treatment were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting. To elucidate how regulation was reflected in biochemical pathways, endogenous metabolites were measured in serum samples collected 48 h post-dosing by 600-MHz ¹H-NMR. In summary, a single dose of hydrazine caused gene, protein and metabolite changes, which can be related to glucose metabolism, lipid metabolism and oxidative stress. These findings support known effects of hydrazine toxicity and provide potential new biomarkers of hydrazine-induced toxicity.     

Phenylphosphonate transport by Helicobacter pylori

BACKGROUND: Helicobacter pylori can utilize phenylphosphonate as a sole source of phosphorus, and it is able to transport the phosphonate N-phosphonoacetyl-L-aspartate. However, H. pylori does not have any genes homologous to those of the known pathways for phosphonate degradation in bacteria, indicating that it must have novel pathways for the transport and metabolism of phosphonates. METHODS: Phenylphosphonate transport by H. pylori was studied in strains LC20, J99 and N6 by the centrifugation through oil method using [(14)C]-labeled phenylphosphonate. RESULTS: The Michaelis constants of transport K(t) and V(max) for phenylphosphonate showed similar kinetics in the three strains. The Arrhenius plot for phenylphosphonate transport rates at permeant concentrations of 50 micromol/L was linear over the temperature range 10-40 degrees C with an activation energy of 3.5 kJ/mol, and a breakpoint between 5 and 10 degrees C. Transport rates increased with monovalent cation size. The effects of various inhibitors were investigated: iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of phenylphosphonate transport; sodium azide and sodium cyanide increased the transport rate; and monensin had no effect. CONCLUSIONS: The kinetics and properties of H. pylori phenylphosphonate transport were characterized, and the data suggested a carrier-mediated transport mechanism.     

蛋白质组学在神经科学中的应用

蛋白质组学是指对基因组编码的所有蛋白质进行大规模分析的一门学科,它分为表达蛋白质组学和功能蛋白质组学。新的蛋白质组学工具将为高度复杂的神经科学的研究提供便利。作者简述了表达蛋白质组学和功能蛋白质组学在这一领域的应用。     

白癜风患者病变表皮与正常表皮的比较蛋白质组学研究

目的 本文通过开展白癜风患者病变表皮与正常表皮的比较蛋白质组学研究,发现和鉴定白癜风患者病变表皮与正常表皮之间的差异表达蛋白,以探讨白癜风患者表皮发生病变的分子机制。方法 首先,建立和优化了表皮样品中蛋白质的最佳酶切条件。其次,采用基于串联质谱标签（TMT）标记的定量蛋白质组学技术策略开展了稳定期白癜风患者病变表皮与正常表皮的比较蛋白质组学研究,并筛选了差异表达蛋白。最后通过生物信息学分析工具及数据库（GO、KEGG、STRING、GSEA）对差异蛋白进行功能富集分析。结果 优化所得到的最佳酶解条件是由Lys-C （酶∶底物,1∶100）和胰酶（酶∶底物,1∶50）组合而成的顺序酶切。比较蛋白质组学研究共鉴定4 496个蛋白质,其中181个蛋白质为白癜风患者病变表皮中的差异表达蛋白。生物信息学分析表明差异表达蛋白主要与代谢、免疫、氧化还原和细胞黏附相关。其中119个上调蛋白主要参与角质化、转录、氧化应激及蛋白酶解等过程。62个下调蛋白主要参与细胞内物质运输、谷胱甘肽代谢和肌动蛋白细丝封端等过程。结论 比较蛋白质组学研究揭示了白癜风患者病变表皮与正常表皮之间主要存在角质化、免疫、脂质代谢...     

Advances and challenges in understanding the role of the lipid raft proteome in human health

ABSTRACT

Introduction: Phase separation as a biophysical principle drives the formation of liquid-ordered ‘lipid raft’ membrane microdomains in cellular membranes, including organelles. Given the critical role of cellular membranes in both compartmentalization and signaling, clarifying the roles of membrane microdomains and their mutual regulation of/by membrane proteins is important in understanding the fundamentals of biology, and has implications for health.

Areas covered: This article will consider the evidence for lateral membrane phase separation in model membranes and organellar membranes, critically evaluate the current methods for lipid raft proteomics and discuss the biomedical implications of lipid rafts.

Expert commentary: Lipid raft homeostasis is perturbed in numerous chronic conditions; hence, understanding the precise roles and regulation of the lipid raft proteome is important for health and medicine. The current technical challenges in performing lipid raft proteomics can be overcome through well-controlled experimental design and careful interpretation. Together with technical developments in mass spectrometry and microscopy, our understanding of lipid raft biology and function will improve through recognition of the similarity between organelle and plasma membrane lipid rafts and considered integration of published lipid raft proteomics data.     

A new,long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.     

体内肉碱来源及其对脂类代谢的影响

肉碱是体内一种有多种生理功能的氨基酸类物质,其在脂类代谢过程中有重要的调节作用,近年来在甘油三脂血症、肾透析患者的脂代谢障碍的辅助治疗中取得了较好疗效。本文就肉碱的来源和对脂类代谢的影响进行了讨论。     

使用BODIPY荧光染料快速测定微藻油脂含量方法

使用BODIPY505/515荧光染料,通过荧光分光光度法测定藻细胞中的油脂含量。结果表明：BODIPY505/515的最佳染色条件为二甲基亚砜（DMSO）体积分数2%,BODIPY505/515最终质量浓度0.25μg/mL,染色时间30min,染色温度35℃。在最佳染色条件下,微藻油脂含量与荧光强度呈线性相关（R2=0.976 4）。通过测定BODIPY505/515染色的不同种属微藻的荧光强度,应用该关系计算其油脂含量,与质量法测定的结果相比没有显著差异。该方法较为普适,比传统方法相比具有简便快捷,试样用量少的特点,与尼罗红荧光染料相比具有较窄的发射波谱范围,不会与微藻的自身荧光相互干扰,更适于过程监控及高含油藻株的筛选。     

A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY)-labeled lipids

A series of fluorescently labeled fatty acids of various chain lengths with 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY-8) residue in the ω-position were synthesized. These acids were used to prepare new fluorescently labeled phosphatidylcholines, sphingomyelin, and galactosyl ceramide. The symmetry of the Me₄-BODIPY-8-fluorophore suggests that, in most bilayer membrane systems, this fluorophore would be embedded into the bilayer.     

SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。