Rapid methods for the characterization of unilamellar phospholipid vesicles. Application to studies on fatty acid donor and acceptor properties of membranes and fatty acid binding proteins

Vesicles having diameters from 20 to 200 nm were prepared from egg-yolk phosphatidylcholine (PC) and were separated as well as analyzed by methods that can be carried out with standard laboratory equipment. Gel-chromatography on Sephacryl S 1000 was adapted for expeditious size analysis of vesicles as well as for isolation of vesicle populations having a narrow range of diameters. The internal volume of vesicles was derived from enzymic tests for PC and for glucose encapsulated. Size analysis and enzymic determinations provided a convenient check for the lamellarity of membranes produced.Fatty acids and fatty acid binding proteins (FABPs) must interact in vivo in the presence of cellular membranes. As a model, interactions between unilamellar vesicles, anthroyloxypalmitic acid (A16:0) and FABPs were studied with the aid of gel-chromatographic methods elaborated and of fluorescence spectroscopy. FABP from bovine heart donated A16:0 to membranes, whereas FABP from bovine liver removed this fatty acid from vesicle membranes. The results revealed characteristic differences between cardiac and hepatic FABPs with regard to binding a fatty acid.     

The glycolipid fatty acids of Porphyridium purpureum cultured in the presence of detergents

The glycolipid fatty acid composition of Porphyridium purpureum on a solid medium was studied in the presence of Triton X-100 (TX), sodium desocycholate (SDC), sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB). TLC and GC/MS were used in determining the relative fatty acid compositions of mono-and digalactosyl diglycerides (MGDG and DGDG) and in assessing the MGDG/DGDG ratio. The most common fatty acids were palmitic (16:0), stearic (18:0), linoleic (18:2 ω6), arachidonic (20:4 ω6) and eicosapentaenoic (20:5 ω3) acids, the long-chain polyunsaturated acids being more abundant in DGDG; α-linolenic acid (18:3 ω3) was absent. TX and SDC in particular caused an increase in the saturation grade of both MGDG and DGDG fatty acids at very low concentrations (5–15 ppm). With a detergent concentration of 20 ppm a reversion of this tendency was sometimes found, and the fatty acid composition approached the controls again. The effects of SDS and CTAB were not as prominent. All the detergents studied increased the normal MGDG/DGDG ratio (0.3) to a maximum of ～ 1. The effect of increasing detergent concentration is not linear. The results suggest that in some cases very low detergent concentrations can be more effective than higher ones, a fact which may be important in algae growing in polluted waters.     

Heparan sulfate proteoglycans and heparin regulate melanoma cell functions

Background

The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions.

Scope of review

This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression.

Major conclusions

We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis.

General significance

The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

脱氧胆酸钠体系分离紫杉烷类化合物

以脱氧胆酸钠(SDC)为手性拆分剂,建立了紫杉醇、10-去乙酰基-紫杉醇、10-去乙酰基-7-表-紫杉醇、三尖杉宁碱、巴可亭Ⅲ、10-去乙酰基-巴可亭Ⅲ、10-去乙酰基-7-表-巴可亭Ⅲ等7种紫杉烷类化合物的胶束电动毛细管色谱分离方法.分离条件为67cm×75μm毛细管,分离电压25kv,温度25℃,UV检测器230nm,气动进样3s.缓冲液为脱氧胆酸50mm,Tris-HCl140mm,MeOH20%,pH8.5.     

Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

Efficient mining differential co-expression biclusters in microarray datasets

Background

Biclustering algorithm can find a number of co-expressed genes under a set of experimental conditions. Recently, differential co-expression bicluster mining has been used to infer the reasonable patterns in two microarray datasets, such as, normal and cancer cells.

Methods

In this paper, we propose an algorithm, DECluster, to mine Differential co-Expression biCluster in two discretized microarray datasets. Firstly, DECluster produces the differential co-expressed genes from each pair of samples in two microarray datasets, and constructs a differential weighted undirected sample–sample relational graph. Secondly, the differential biclusters are generated in the above differential weighted undirected sample–sample relational graph. In order to mine maximal differential co-expression biclusters efficiently, we design several pruning techniques for generating maximal biclusters without candidate maintenance.

Results

The experimental results show that our algorithm is more efficient than existing methods. The performance of DECluster is evaluated by empirical p-value and gene ontology, the results show that our algorithm can find more statistically significant and biological differential co-expression biclusters than other algorithms.

Conclusions

Our proposed algorithm can find more statistically significant and biological biclusters in two microarray datasets than the other two algorithms.     

Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins

Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.     

Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography–tandem mass spectrometry (LC–MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS^E) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC–MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.     

不同病情老年社区获得性肺炎患者血清ALB、NT-proBNP、PTX3、SDC4的变化及对预后的影响分析

摘要 目的：分析不同病情老年社区获得性肺炎（CAP）患者血清白蛋白（ALB）、N末端脑钠肽前体（NT-proBNP）、正五聚蛋白3（PTX3）、多配体蛋白聚糖4（SDC4）的变化并探讨其对患者预后的影响。方法：选取2019年1月～2020年1月西南医科大学附属医院收治的196例老年CAP患者,根据病情严重程度分为重症CAP组80例和非重症CAP组116例,根据入院后28 d存活情况分为死亡组25例和存活组171例。收集患者临床资料,检测血清ALB、NT-proBNP、PTX3、SDC4水平。通过多因素Logistic回归分析老年CAP患者死亡的影响因素,受试者工作特征（ROC）曲线分析血清ALB、NT-proBNP、PTX3、SDC4水平对老年CAP患者死亡风险的预测价值。结果：与非重症CAP组比较,重症CAP组血清ALB、SDC4水平降低,NT-proBNP、PTX3水平升高（P＜0.05）。多因素Logistic回归分析显示,ALB升高、SDC4升高为老年CAP患者死亡的保护因素,呼吸频率加快、CURB-65评分增加、肺炎严重指数（PSI）增加、NT-proBNP升高、PTX3升高为老年CAP患者死亡的危险因素（P＜0.05）。ROC曲线分析显示,血清ALB、NT-proBNP、PTX3、SDC4水平联合预测老年CAP患者死亡的曲线下面积（AUC）大于各指标单独预测。结论：老年CAP患者体内血清ALB、SDC4水平降低,NT-proBNP、PTX3水平升高,血清ALB、NT-proBNP、PTX3、SDC4与患者病情加重和预后有关,具有作为老年CAP患者预后评估指标的潜能。     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.