Analysis of fatty acids in A. szechenyianum Gay. by microwave‐assisted extraction and gas chromatography‐mass spectrometry

Introduction – Aconitum szechenyianum Gay. is a traditional Chinese medicinal herb with the detumescent and styptic effects and antitumor activity. There have been only a few researches on its chemical components, but no detailed report has appeared on its fatty acids. Objective – To develop a simple and effective method for the extraction of fatty acids from A. zechenyianum Gay. and then to investigate the fatty acid components. Methodology – Microwave‐assisted extraction (MAE) was optimized with response surface methodology, and the fatty acid compositions of extract were determined by GC–MS with previous derivatisation to fatty acid methyl esters (FAMEs). The results were compared with that obtained by classical Soxhlet extraction (SE). Results – Compared with SE, MAE showed significantly higher fatty acid yields, shorter extraction time, and lower energy and solvent consumption. The major fatty acids in A. szechenyianum Gay. are linoleic acid, palmitic acid, linolenic acid, oleic acid and stearic acid, and the unsaturated fatty acids occupy 66.4% of the total fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

The Chemiluminescence Assay of Lipid Peroxidation Products in Human Blood Plasma Lipoproteins

A chemiluminescence (CL) flash kinetics on the addition of Fe²⁺ ions into oxidized low density lipoprotein (LDL) suspension has been studied. LDL oxidation was carried out at 37°C without and in the presence of 5 or 50 μM of Cu.²⁺ It has been found that under certain experimental conditions (the addition of excess iron ions, more than 1 mM) the amplitude of CL flash depended almost linearly (1) on the concentration of oxidized LDL and (2) on the extent of LDL oxidation measured as diene conjugates (DC) and 2-thiobarbituric acid-reactive substance (TBARS) accumulation. The corresponding correlation coefficients were: for TBARS - 0.94 and for DC - 0.97, in the case of LDL autooxidation; 0.72 and 0.98, in the case of copper-induced LDL oxidation. A sensitivity of the CL method was shown to be significantly enhanced (by more than two orders) in the presence of CL sensitizer - 2, 3,5, 6-lH,4H-tetrahydro-9-(2' -benzoimidazolyl)-quinolizin-(9, 9a, 1 -gh)coumarin.     

Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins.     

基于主成分分析法探讨人体测量学参数与青少年血糖关系的研究

目的:调查分析人体测量学参数包括体重指数(BMI)、腰围(WC)、臀围(HC)、腰围/臀围比(WHR)及腰围/身高比(WHtR)对南昌地区青少年空腹血糖的影响。方法:在南昌地区进行随机抽样检查731例12-18岁之间的青少年,测量身高、体重、BMI、WC、HC、WHR、WHtR和空腹血糖,用SAS软件进行统计分析。结果:男女性的五项人体测量学指标和空腹血糖间均呈显著正相关,相关系数为0.14-0.38;通过主成分分析得到四个主成分,多重回归分析结果表明联合四个主成分比单独使用其中任何一个更能解释空腹血糖的变异。结论:BMI、WC、HC、WHR、WHtR与男性和女性空腹血糖都有一定相关性,并且联合这五项测量学指标比单独使用其中任何一个能更精确预测空腹血糖。     

Arv1 Regulates PM and ER Membrane Structure and Homeostasis But is Dispensable for Intracellular Sterol Transport

The pan‐eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild‐type and Arv1‐deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild‐type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C‐terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail‐anchored proteins involved in membrane homoeostasis .     

Essential trace elements in humans

Serum arsenic concentrations of persons suffering from renal failure and undergoing hemodialysis treatment (n=85) and of healthy controls (n=25) were determined by hydride-generation AAS technique after microwave digestion. The results were evaluated by comparing the values of both groups, considering physiological factors and individual data, as well as comorbid conditions of the hemodialysis (HD) patients. Serum arsenic levels were diminished in the patient group compared with controls (mean values 8.5±1.8 ng/mL vs 10.6±1.3 ng/mL). Furthermore, additional diseases within the hemodialysis group, particularly injuries of the central nervous system (CNS), vascular diseases, and cancer, were correlated to occasionally markedly decreased serum arsenic concentrations. It was concluded that arsenic homeostasis is disturbed by HD treatment and certain additional diseases. Desirable arsenic concentrations in the body seem to be reasonable. This consideration results in the conclusion that arsenic could play an essential role in human health. Thus, reference arsenic concentrations in different human tissues and body fluids should be established in order to recognize not only arsenic intoxication, but also arsenic deficiency. Perhaps arsenic deficiency contributes to the increased death risk of HD patients, and therefore, arsenic supplementations for patients with extremely low serum arsenic concentrations should be taken into account.     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) lumen triggers the opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca²⁺ store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.     

Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca^2 + concentration. The oligomerization of STIM1, which triggers extracellular Ca^2 + influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca^2 + concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca^2 + concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca^2 + concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca^2 + influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca^2 + loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca^2 +-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.