脂肪酸对人肺腺癌细胞膜流动性的影响

脂肪酸是细胞膜正常流动性的主要调节因素之一。本文报导了二种不同转移表型人肺腺细胞与九种不同脂肪酸共孵育后,对其细胞膜流动性的影响。结果表明,不同转移一夫肺腺癌细胞对各种脂肪酸有不同的敏感性,高转移癌细胞Ａｎｉｐ对棕榈酸和花生酸较敏感,而低转移癌细胞ＡＧＺＹ对棕榈烯酸和亚油酸较敏感。     

高寒山区牧草根质膜和脂肪酸组分对冷冻低温的适应反应

在秋末冬初测定了高寒山区自然生境中生长的垂穗披碱草(Elymus nutans)、早熟禾(Poa sphyondylodes) 和花雀麦(Bromus sinensis)3种牧草根膜脂脂肪酸组分和质膜流动性及ATPase活力。结果表明：随着秋末冬初气温下降到0℃以下,3种牧草根中膜脂肪酸配比发生较大变化,棕榈酸相对百分含量下降40.29％,亚油酸增加3倍多,亚麻酸增加112.39％,脂肪酸不饱和指数(IUFA)增大;同时根质膜流动性在低温期间较大,在融冻阶段有下降,冰冻阶段增大;质膜Mn2+ATPase、Ca2+ ATPase活力增大,Mg2+ ATPase活力下降。在冷冻适应中牧草根膜脂脂肪酸不饱和度的增加直接影响着膜的流动性,影响膜功能和植物抗冻性,并在维持根细胞膜完整性和抵抗组织结冰伤害方面起重要作用。     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.     

Effects of weight loss on erythrocyte membrane composition and fluidity in overweight and moderately obese women

A previous study showed chemical and physical impairment of the erythrocyte membrane of overweight and moderately obese women. The present study investigated the effects of a low-calorie diet (800 kcal/day deficit for 8 weeks) on erythrocyte membrane properties in 70 overweight and moderately obese (body mass index, 25-33 kg/m²) normotensive, nondiabetic women. At the end of dietary intervention, 24.3% of women dropped out, 45.7% lost less than 5% of their initial weight (Group I) and only 30% of patients lost at least 5% of their initial body weight (Group II). Group I showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of Group II showed significant reductions in malondialdehyde, lipofuscin, cholesterol, sphingomyelin, palmitic acid and nervonic acid and an increase in di-homo-γ-linolenic acid, arachidonic acid and membrane fluidity. Moreover, Group II showed an improvement in total cholesterol, low-density lipoprotein cholesterol, glycemia and insulin resistance. These changes in erythrocyte membrane composition could reflect a virtuous cycle resulting from the reduction in insulin resistance associated with increased membrane fluidity that, in turn, results in a sequence of metabolic events that concur to further improve membrane fluidity.     

脂肪族类两亲物对肌浆网膜脂区结构的影响

C18饱和脂肪酸和胺可增加DPH标记肌浆网(SR)的荧光偏振度,而C18单不饱和脂肪酸。胺和醇则使其偏振度下降。加入MgATP,可除去单不饱和脂肪胺引起的DPH标记的荧光偏振度下降,并使之高于未加脂肪胺的对照水平。饱和酸及相应胺可使标记于膜脂中层和深层的TAS和12AS的荧光偏振度上升,不饱和酸及相应胺和醇仅使12AS荧光偏振下降。说明脂肪族类两亲物对SR膜流动性的影响与脂肪链饱和程度有关。饱和者主要使膜中、深层流动性下降.不饱和者主要使膜深层流动性升高。     

Changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of Neurospora crassa

Growth of a choline requiring auxotroph of Neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. Whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. In these lipids, choline deprivation produced little change in fatty acid profiles of phosphatidylethanolamine, whereas changes in fatty acids of phosphatidylcholine and acidic phospholipids resulted in increased levels of unsaturation at both growth temperatures. Microsomal phospholipids also showed fatty acid variability with sharp decreases in phosphatidylcholine unsaturates and increases in acidic phospholipid unsaturated fatty acids at low growth temperatures. Fluorescence polarization of 1,6-diphenylhexatriene in vesicles formed from total cellular and microsomal lipids showed that choline deprivation produces changes in thermotropic properties in the lipids in deprived cultures at either growth temperature. The effective differences in fluorescence polarization between choline-deprived and supplemented cultures grown at a given temperature were found to be comparable to those produced by temperature acclimation in normally growing cultures over a temperature range of 22 K.     

Fibril formation from cellulose by a novel protein from Trichoderma reesei: A non-hydrolytic cellulolytic component?

A low molecular weight protein, named fibril-forming protein (FFP), was isolated from the culture supernatant of Avicel-grown Trichoderma reesei. The protein was purified to homogeneity and it exhibited a molecular weight of 11,400Da. Low amounts of this protein caused apparently non-hydrolytic disruption of filter paper, releasing fibrils without any detectable release of reducing sugars. It displayed no hydrolytic activity on carboxymethylcellulose (CMC), p-nitrophenyl--d-glucoside (pNPG) or 4-methylumbelliferyl cellobioside. The pH optimum of the protein was between 4 and 5. The temperature optimum was 40°C and the computed activation energy (E_a) for the filter paper disruption process was 4.18kcal/mol, suggesting disruption of non-covalent bonds. It had no immunological cross reactivity with reported cellulase components of T. reesei.     

Lipid Composition and Temperature Adaptation of the Nervous System of the Leech Hirudo medicinalis L.

The lipid composition of the nervous system of the leech Hirudo medicinalis was investigated following acclimatization of animals at 25 degrees C and 5 degrees C. Choline, ethanolamine, and serine plus inositol phosphoglycerides are the major phospholipid classes of the leech ganglionic chain; minor amounts of lysophosphatidylcholine, phosphatidic acid, and sphingomyelin are also present. Neither the phospholipid pattern nor the cholesterol to total phospholipid molar ratio was dependent on the acclimatization temperature, whereas the fatty acid patterns of choline and serine plus inositol phosphoglycerides were significantly affected. Both for choline and serine plus inositol phosphoglycerides, a significant increase of the unsaturation index and a decrease of saturated to unsaturated fatty acid ratio was observed in animals acclimatized at 5 degrees C in comparison with those acclimatized at 25 degrees C. These observations, which point to increased lipid fluidity of the nervous system of cold-adapted leeches, are strengthened by results obtained by the fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene as a probe: a decrease of the fluorescence polarization value was observed throughout the temperature range selected (5-40 degrees C).     

The effect of natural and synthetic fatty acids on membrane structure,microdomain organization,cellular functions and human health

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Proteomics-based consensus prediction of protein retention in a bacterial membrane

The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins that are potentially retained in the cytoplasmic membranes of the corresponding organisms. In practice, however, major problems are encountered with the computer-assisted distinction between (Sec-type) signal peptides that direct exported proteins into the growth medium and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. In the present studies, which were aimed at improving methods to predict protein retention in the bacterial cytoplasmic membrane, we have compared sets of membrane-attached and extracellular proteins of Bacillus subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins can be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. This unprecedented finding indicates that unknown mechanisms are involved in membrane retention of this class of proteins. A further novelty is a consensus sequence indicative for release of certain lipoproteins from the membrane by proteolytic shaving. Finally, using non-overlapping sets of secreted and membrane-retained proteins, the accuracy of different signal peptide prediction algorithms was assessed. Accuracy for the prediction of protein retention in the membrane was increased to 82% using a majority-vote approach. Our findings provide important leads for future identification of surface proteins from pathogenic bacteria, which are attractive candidate infection markers and potential targets for drugs or vaccines.     

里氏木霉液体发酵产纤维素酶的研究

在摇瓶试验基础上,采用里氏木霉(Trichoderma reesei)HC-415菌株进行5L自控罐产纤维素酶深层发酵试验。在通气量为 0.2—0.6vvm、搅拌速度为 400r/min、发酵液pH控制在5.8—6.1的条件下,发酵液的羧甲基纤维素(CMC)酶酶活最高为325.0mg糖/ml,滤纸糖酶(FPA)酶活最高达17.9mg糖/ml。发酵周期为108h。所得冻干纤维素酶粉CMC酶活最高3111IU/g,FPA最高135IU/g ,对发酵液得率平均6.7g/L。酶活总收率CMC酶活平均78.2％,FPA酶活平均73.5％。     

Pancreatic islet response to hyperglycemia is dependent on peroxisome proliferator-activated receptor alpha (PPARalpha)

This study tests the hypothesis that islet peroxisome proliferator-activated receptor alpha (PPARalpha) influences insulin secretion. Freshly isolated islets of normoglycemic PPARalpha-null mice display no major alteration of glucose-stimulated insulin release. However, after 24 h of culture in high glucose, PPARalpha-null islets exhibit elevated basal insulin secretion and fail to increase insulin mRNA. 24-h culture with palmitate replicates this phenotype in wild-type islets. The data suggest that PPARalpha is needed to ensure appropriate insulin secretory response in situation of short-term hyperglycemia, likely by maintaining islet lipid homeostasis. As such, islet PPARalpha could contribute to delay the progression of type 2 diabetes.     

Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30

Trichoderma reesei was grown in a stirred-tank bioreactor (STB) and a reciprocating plate bioreactor (RPB) at four different agitation speeds. A semiautomatic image analysis protocol that was developed to characterize the mycelium morphology during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms) was applied to study the effect of agitation on the morphology of T. reesei. It was shown via statistical validation that broth samples used for image analysis represented the whole population of the fungi in the bioreactor. High shear was found to be damaging to T. reesei grown in the STB. The gentler shear produced in the RPB was not detrimental to the microorganism even at higher agitation speed. Better productivity was obtained for T. reesei grown in the STB and the highest productivity, 0.121 IU/mL h, was obtained at 400 rpm. The morphological parameter, the hyphal growth unit, was found to be correlated to the productivity. Understanding the effect of agitation on the morphology and productivity of T. reesei could lead to the design of better bioreactors and the selection of operating conditions of bioreactors to optimize the production of cellulase.     

Identification of Key Residues and Regions Important for Porcupine-mediated Wnt Acylation

Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.